Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell

ABSTRACT

Described herein are methods for identifying nucleic acid sequences that modulate the function of a cell, the expression of a gene in a cell, or the biological activity of a target polypeptide in a cell. The methods involve the use of double stranded RNA expression libraries, double stranded RNA molecules, and post-transcriptional gene silencing techniques.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of the filing date of U.S.provisional applications Nos. 60/265,805, filed Jan. 31, 2001, and60/339,260, filed Oct. 26, 2001.

BACKGROUND OF THE INVENTION

[0002] The invention relates to methods for identifying nucleic acidsequences that modulate the function of a cell, by the use ofpost-transcriptional gene silencing.

[0003] Double stranded RNA (dsRNA) has been shown to inducesequence-specific gene silencing in a number of different organisms.Gene silencing can occur through various mechanisms, one of which ispost-transcriptional gene silencing (PTGS). In post-transcriptional genesilencing, transcription of the target locus is not affected, but theRNA half-life is decreased. The mechanisms by which PTGS occurs are notyet clear. Exogenous dsRNA has been shown to act as a potent inducer ofPTGS in nematodes, trypanosomes, and insects. In addition, studies in C.elegans and Drosophila show that a few molecules of dsRNA per cell aresufficient to trigger a PTGS response. Furthermore, studies in mice havedemonstrated that dsRNA can interfere with the expression of genes inmouse embryos.

[0004] There exists a need to identify molecules that selectivelyregulate the expression of genes in vertebrate cells without theassociated toxicity of the interferon response. Such regulation shouldallow the downregulation of expression from genes whose gene productsare detrimental to the cells.

SUMMARY OF THE INVENTION

[0005] In general the invention features high throughput methods ofusing PTGS to identify a nucleic acid sequence that modulates thefunction of a cell, gene expression of a target nucleic acid, or thebiological activity of a target polypeptide. The method involves the useof specially constructed cDNA libraries derived from a cell, forexample, a primary cell or a cell line that has an observable phenotypeor biological activity, (e.g., an activity mediated by a targetpolypeptide or altered gene expression), that are transfected into cellsto inhibit gene expression. This inhibition of gene expression altersthe function of a cell, gene expression of a target nucleic acid, or thebiological activity of a target polypeptide, and the nucleic acidsequence responsible for the modulation can be readily identified. Themethod may also utilize randomized nucleic acid sequences or a givensequence for which the function is not known. Although the use of PTGSas a validation strategy is known in the art, its use in screeningtechniques, as described herein, is novel.

[0006] Accordingly, in a first aspect, the invention features a methodfor identifying a nucleic acid sequence that modulates the function of acell. The method involves: (a) transforming a population of cells with adouble stranded RNA expression library, where the library is derivedfrom the cells, where at least two cells of the population of cells areeach transformed with a different nucleic acid from the double strandedRNA expression library, and where the nucleic acid is capable of formingdouble stranded RNA; (b) optionally selecting for a cell in which thenucleic acid is expressed in the cell; and (c) assaying for a modulationin the function of the cell, wherein a modulation identifies a nucleicacid sequence that modulates the function of a cell.

[0007] In a desirable embodiment of the first aspect of the invention,assaying for a modulation in the function of a cell comprises measuringcell motility, apoptosis, cell growth, cell invasion, vascularization,cell cycle events, cell differentiation, cell dedifferentiation,neuronal cell regeneration, or the ability of a cell to support viralreplication.

[0008] In a second aspect, the invention features a method foridentifying a nucleic acid sequence that modulates expression of atarget nucleic acid in a cell. The method involves: (a) transforming apopulation of cells with a double stranded RNA expression library, wherethe library is derived from the cells, where at least two cells of thepopulation of cells are each transformed with a different nucleic acidfrom the double stranded RNA expression library, and where the nucleicacid is capable of forming double stranded RNA; (b) optionally selectingfor a cell in which the nucleic acid is expressed in the cell; and (c)assaying for a modulation in the expression of a gene in the cell, wherea modulation identifies a nucleic acid sequence that modulatesexpression of a target nucleic acid in a cell.

[0009] In a desirable embodiment of the second aspect of the invention,the target nucleic acid is assayed using DNA array technology.

[0010] In a third aspect, the invention features a method foridentifying a nucleic acid sequence that modulates the biologicalactivity of a target polypeptide in a cell. The method involves: (a)transforming a population of cells with a double stranded RNA expressionlibrary, where the library is derived from the cells, where at least twocells of the population of cells are each transformed with a differentnucleic acid from the double stranded RNA expression library, and wherethe nucleic acid is capable of forming double stranded RNA;(b)optionally selecting for a cell in which the nucleic acid isexpressed in the cell; and (c) assaying for a modulation in thebiological activity of a target polypeptide in the cell, wherein amodulation identifies a nucleic acid sequence that modulates thebiological activity of a target polypeptide.

[0011] In one embodiment of any of the above aspects of the invention,in transforming step (a), the nucleic acid is stably integrated into achromosome of the cell. Integration of the nucleic acid may be random orsite-specific. Desirably integration is mediated by recombination orretroviral insertion. In addition, desirably a single copy of thenucleic acid is integrated into the chromosome. In another embodiment ofany of the above aspects of the invention, in step (a) at least 50, moredesirably 100; 500; 1000; 10,000; or 50,000 cells of the population ofcells are each transformed with a different nucleic acid from the doublestranded RNA expression library. In other embodiments, the population ofcells is transformed with at least 5%, more desirably at least 25%, 50%,75%, or 90%, and most desirably at least 95% of the double stranded RNAexpression library. In yet another embodiment, the method furtherinvolves: (d) identifying the nucleic acid sequence by amplifying andcloning the sequence. Desirably amplification of the sequence involvesthe use of the polymerase chain reaction (PCR).

[0012] In other embodiments of any of the above aspects of theinvention, the double stranded RNA expression library contains cDNAs orrandomized nucleic acids. The double stranded RNA expression library maybe a nuclear double stranded RNA expression library, in which case thedouble stranded nucleic acid is made in the nucleus. Alternatively, thedouble stranded RNA expression library may be a cytoplasmic doublestranded RNA expression library, in which case the double strandednucleic acid is made in the cytoplasm. In addition, the nucleic acidfrom the double stranded RNA expression library may be made in vitro orin vivo. In addition, the identified nucleic acid sequence may belocated in the cytoplasm of the cell.

[0013] In still another embodiment of any of the above aspects of theinvention, the nucleic acid is contained in a vector, for example adouble stranded RNA expression vector. The vector may then betransformed such that it is stably integrated into a chromosome of thecell, or it may function as an episomal (non-integrated) expressionvector within the cell. In one embodiment, a vector that is integratedinto a chromosome of the cell contains a promoter operably linked to anucleic acid encoding a hairpin or double stranded RNA. In anotherembodiment, the vector does not contain a promoter operably linked to anucleic acid encoding a double stranded RNA. In this later embodiment,the vector integrates into a chromosome of a cell such that anendogenous promoter is operably linked to a nucleic acid from the vectorthat encodes a double stranded RNA. Desirably, the double stranded RNAexpression vector comprises at least one RNA polymerase II promoter, forexample, a human CMV-immediate early promoter (HCMV-IE) or a simian CMV(SCMV) promoter, at least one RNA polymerase I promoter, or at least oneRNA polymerase III promoter. The promoter may also be a T7 promoter, inwhich case, the cell further comprises T7 polymerase. Alternatively, thepromoter may be an SP6 promoter, in which case, the cell furthercomprises SP6 polymerase. The promoter may also be one convergent T7promoter and one convergent SP6 promoter. A cell may be made to containT7 or SP6 polymerase by transforming the cell with a T7 polymerase or anSP6 polymerase expression plasmid, respectively. In some embodiments, aT7 promoter or a RNA polymerase III promoter is operably linked to anucleic acid that encodes a small double stranded RNA (e.g., a doublestranded RNA that is less than 200, 150, 100, 75, 50, or 25 nucleotidesin length). In other embodiments, the promoter is a mitochondrialpromoter that allows cytoplasmic transcription of the nucleic acid inthe vector (see, for example, the mitochondrial promoters described inWO 00/63364, filed Apr. 19, 2000). Alternatively, the promoter is aninducible promoter, such as a lac (Cronin et al. Genes & Development 15:1506-1517, 2001), ara (Khlebnikov et al, J Bacteriol. 2000 December;182(24):7029-34), ecdysone (Rheogene, www.rheogene.com), RU48(mefepristone) (corticosteroid antagonist) (Wang X J, Liefer K M, TsaiS, O'Malley B W, Roop D R, Proc Natl Acad Sci U S A. 1999 July20;96(15):8483-8), or tet promoter (Rendal et al., Hum Gene Ther. 2002January; 13(2):335-42. and Larnartina et al., Hum Gene Ther. 2002January;13(2):199-210) or a promoter disclosed in WO 00/63364, filedApr. 19, 2000. In desirable embodiments, the inducible promoter is notinduced until all the episomal vectors are eliminated from the cell. Thevector may also comprise a selectable marker. In addition, these vectorsmay be used in combination with methods that inhibit or prevent aninterferon response or double stranded RNA stress response, as describedherein.

[0014] Desirably in a vector for use in any of the above aspects of theinvention, the sense strand and the antisense strand of the nucleic acidsequence are transcribed from the same nucleic acid sequence using twoconvergent promoters. In another desirable embodiment, in a vector foruse in any of the above aspects of the invention, the nucleic acidsequence comprises an inverted repeat, such that upon transcription, thenucleic acid forms a double stranded RNA.

[0015] In still other embodiments of any of the above aspects of theinvention, the cell and the vector each further comprise a loxP site andsite-specific integration of the nucleic acid into a chromosome of thecell occurs through recombination between the loxP sites. In addition,step (b) of any of the above aspects of the invention further involvesrescuing the nucleic acid through Cre-mediated double recombination.

[0016] In still further embodiments of any of the above aspects of theinvention, the identified nucleic acid sequence is located in thenucleus of the cell. Alternatively, the identified nucleic acid sequencemay be located in the cytoplasm of the cell.

[0017] In yet another embodiment of any of the above aspects of theinvention, the nucleic acid from the double stranded RNA expressionlibrary is at least 100, 500, 600, or 1000 nucleotides in length. Inother embodiments of any of the above aspects of the invention, thenucleic acid from the double stranded RNA expression library is at least10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In yetother embodiments, the number of nucleotides in the nucleic acid fromthe double stranded RNA expression library is between 5-100 nucleotides,15-100 nucleotides, 20-95 nucleotides, 25-90 nucleotides, 35-85nucleotides, 45-80 nucleotides, 50-75 nucleotides, or 55-70 nucleotides,inclusive. In still other embodiments, the number of nucleotides in thenucleic acid from the double stranded RNA expression library iscontained in one of the following ranges: 5-15 nucleotides, 15-20nucleotides, 20-25 nucleotides, 25-35 nucleotides, 35-45 nucleotides,45-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90nucleotides, or 90-100 nucleotides, inclusive. In other embodiments, thenucleic acid contains less than 50,000; 10,000; 5,000; or 2,000nucleotides. In addition, the nucleic acid from the double stranded RNAexpression library may contain a sequence that is less than a fulllength RNA sequence.

[0018] In still further embodiments of any of the above aspects of theinvention, the cell is a plant cell or an animal cell. Desirably theanimal cell is a vertebrate or mammalian cell, for example, a humancell. The cell may be ex vivo or in vivo. The cell may be a gamete or asomatic cell, for example, a cancer cell, a stem cell, a cell of theimmune system, a neuronal cell, a muscle cell, or an adipocyte.

[0019] Transformation/transfection of the cell may occur through avariety of means including, but not limited to, lipofection,DEAE-dextran-mediated transfection, microinjection, protoplast fusion,calcium phosphate precipitation, viral or retroviral delivery,electroporation, or biolistic transformation. The RNA or RNA expressionvector (DNA) may be naked RNA or DNA or local anesthetic complexed RNAor DNA (Pachuk et al., Biochim. Biophys. Acta 1468:20-30, 2000). Inanother embodiment, the cell is not a C. elegans cell. Desirably thevertebrate or mammalian cell has been cultured for only a small numberof passages (e.g., less than 30 passages of a cell line that has beendirectly obtained from American Type Culture Collection), or are primarycells. Desirably, the vertebrate or mammalian cell is transformed withnucleic acids that are not complexed with cationic lipids.

[0020] In yet another embodiment of any of the above aspects of theinvention, the cell is derived from a parent cell, and is generated by:(a) transforming a population of parent cells with a bicistronic plasmidexpressing a selectable marker and a reporter gene, and comprising aloxP site; (b) selecting for a cell in which the plasmid is stablyintegrated; and (c) selecting for a cell in which one copy of theplasmid is stably integrated in a transcriptionally active locus.Desirably the selectable marker is G418 and the reporter gene is greenfluorescent protein (GFP).

[0021] In still another embodiment of the above aspects of theinvention, generation of the double stranded expression librarycomprises: (a) isolating RNA from a cell; (b) synthesizing cDNAs fromthe RNA of step (a); and (c) cloning each cDNA into a vector. DesirablycDNA synthesis is optimized and/or size selected for the generationand/or selection of cDNAs that are at least 100, 500, 600, or 1000nucleotides in length. In other embodiments, the cDNAs are least 10, 20,30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In yet otherembodiments, the number of nucleotides in the cDNAs is between 5-100nucleotides, 15-100 nucleotides, 20-95 nucleotides, 25-90 nucleotides,35-85 nucleotides, 45-80 nucleotides, 50-75 nucleotides, or 55-70nucleotides, inclusive. In still other embodiments, the number ofnucleotides in the cDNAs is contained in one of the following ranges:5-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-35nucleotides, 35-45 nucleotides, 45-60 nucleotides, 60-70 nucleotides,70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides, inclusive.In other embodiments, the cDNAs contain less than 50,000; 10,000; 5,000;or 2,000 nucleotides. In addition, the cDNA may encode an RNA fragmentthat is less than full length. Desirably the vector comprises twoconvergent T7 promoters, two convergent SP6 promoters, or one convergentT7 promoter and one convergent SP6 promoter, a selectable marker, and/ora loxP site.

[0022] In an additional embodiment of any of the above aspects of theinvention, the method is carried out under conditions that inhibit orprevent an interferon response or double stranded RNA stress response.

[0023] In a fourth aspect, the invention features a method foridentifying a nucleic acid sequence that modulates the function of acell, involving: (a) transforming a population of cells with a doublestranded RNA that is derived from the cells; (b) optionally selectingfor a cell in which the nucleic acid is expressed; and (c) assaying fora modulation in the function of the cell, wherein the modulationidentifies a nucleic acid sequence that modulates the function of acell, wherein the method is desirably carried out under conditions thatinhibit or prevent an interferon response or double stranded RNA stressresponse.

[0024] In a desirable embodiment of the fourth aspect of the invention,assaying for a modulation in the function of a cell comprises measuringcell motility, apoptosis, cell growth, cell invasion, vascularization,cell cycle events, cell differentiation, cell dedifferentiation,neuronal cell regeneration, or the ability of a cell to support viralreplication.

[0025] In a fifth aspect, the invention features a method foridentifying a nucleic acid sequence that modulates expression of atarget nucleic acid in a cell, involving: (a) transforming a populationof cells with a double stranded RNA that is derived from the cells; (b)optionally selecting for a cell in which the nucleic acid is expressed;and (c) assaying for a modulation in the expression of the gene in thecell, wherein the modulation identifies a nucleic acid sequence thatmodulates expression of a target nucleic acid in a cell, wherein themethod is desirably carried out under conditions that inhibit or preventan interferon response or double stranded RNA stress response.

[0026] In a desirable embodiment of the fifth aspect of the invention,the target nucleic acid is assayed using DNA array technology.

[0027] In a sixth aspect, the invention features a method foridentifying a nucleic acid sequence that modulates the biologicalactivity of a target polypeptide in a cell, involving: (a) transforminga population of cells with a double stranded RNA that is derived fromthe cells; (b) optionally selecting for a cell in which the nucleic acidis expressed in the cell; and (c) assaying for a modulation in thebiological activity of a target polypeptide in the cell, wherein themodulation identifies a nucleic acid sequence that modulates thebiological activity of a target polypeptide in a cell, wherein themethod is desirably carried out under conditions that inhibit or preventan interferon response or double stranded RNA stress response.

[0028] In a seventh aspect, the invention features a method foridentifying a nucleic acid sequence that modulates the function of acell, involving: (a) transforming a population of cells with a doublestranded RNA; (b)optionally selecting for a cell in which the nucleicacid is expressed; and (c) assaying for a modulation in the function ofthe cell. Desirably, the modulation identifies a nucleic acid sequencethat modulates the function of a cell, wherein the method is desirablycarried out under conditions that or prevent an interferon response ordouble stranded RNA stress response.

[0029] In a desirable embodiment of the seventh aspect of the invention,assaying for a modulation in the function of a cell comprises measuringcell motility, apoptosis, cell growth, cell invasion, vascularization,cell cycle events, cell differentiation, cell dedifferentiation,neuronal cell regeneration, or the ability of a cell to support viralreplication.

[0030] In a eighth aspect, the invention features a method foridentifying a nucleic acid sequence that modulates expression of atarget nucleic acid in a cell, involving: (a) transforming a populationof cells with a double stranded RNA; (b) optionally selecting for a cellin which the nucleic acid is expressed; and (c) assaying for amodulation in the expression of the gene in the cell, wherein themodulation identifies a nucleic acid sequence that modulates expressionof a target nucleic acid in a cell. Desirably, the method is carried outunder conditions that inhibit or prevent an interferon response ordouble stranded RNA stress response.

[0031] In a desirable embodiment of the eighth aspect of the invention,the target nucleic acid is assayed using DNA array technology.

[0032] In a ninth aspect, the invention features a method foridentifying a nucleic acid sequence that modulates the biologicalactivity of a target polypeptide in a cell, involving: (a) transforminga population of cells with a double stranded RNA; (b) optionallyselecting for a cell in which the nucleic acid is expressed in the cell;and (c) assaying for a modulation in the biological activity of a targetpolypeptide in the cell, wherein the modulation identifies a nucleicacid sequence that modulates the biological activity of a targetpolypeptide in a cell. Desirably, the method is carried out underconditions that inhibit or prevent an interferon response doublestranded RNA stress response.

[0033] In one embodiment of any of the above aspects of the invention,in step (a) at least 2, more desirably 50; 100; 500; 1000; 10,000; or50,000 cells of the population of cells are each transformed with adifferent double stranded RNA from a double stranded RNA expressionlibrary. Desirably, at most one double stranded RNA is inserted intoeach cell. In other embodiments, the population of cells is transformedwith at least 5%, more desirably at least 25%, 50%, 75%, or 90%, andmost desirably, at least 95% of the double stranded RNA expressionlibrary. In still another embodiment of any of the fourth, fifth, orsixth aspects of the invention, the method further involves: (d)identifying the nucleic acid sequence by amplifying and cloning thesequence. Desirably amplification of the sequence involves the use ofthe polymerase chain reaction (PCR).

[0034] In a tenth aspect, the invention features a cell or a populationof cells that expresses a double stranded RNA that (i) modulates afunction of the cell, (ii) modulates the expression of a target nucleicacid (e.g., an endogenous or pathogen gene) in the cell, and/or (iii)modulates the biological activity of a target protein (e.g., anendogenous or pathogen protein) in the cell. Desirably, the cellcontains only one molecular species of double stranded RNA or only onecopy of a double stranded RNA expression vector (e.g., a stablyintegrated vector). Desirably, the cell or population of cells isproduced using one or more methods of the invention. In otherembodiments, the double stranded RNA is expressed under conditions thatinhibit or prevent an interferon response or a double stranded RNAstress response.

[0035] In other embodiments of any of the fourth, fifth, sixth, seventh,eighth, ninth, or tenth aspects of the invention, the double strandedRNA is derived from cDNAs or randomized nucleic acids. In addition, thedouble stranded RNA may be a cytoplasmic double stranded RNA, in whichcase the double stranded nucleic acid is made in the cytoplasm. Thedouble stranded RNA may be made in vitro or in vivo. In addition, theidentified nucleic acid sequence may be located in the cytoplasm of thecell.

[0036] In still another embodiment of any of the fourth, fifth, sixth,seventh, eighth, ninth, or tenth aspects of the invention, the nucleicacid is contained in a vector, for example, a double stranded RNAexpression vector that is capable of forming a double stranded RNA.Desirably the double stranded RNA expression vector comprises at leastone promoter. The promoter may be a T7 promoter, in which case, the cellfurther comprises T7 polymerase. Alternatively, the promoter may be anSP6 promoter, in which case, the cell further comprises SP6 polymerase.The promoter may also be one convergent T7 promoter and one convergentSP6 promoter. A cell may be made to contain T7 or SP6 polymerase bytransforming the cell with a T7 polymerase or an SP6 polymeraseexpression plasmid, respectively. The vector may also comprise aselectable marker, for example hygromycin.

[0037] Desirably in a vector for use in any of the fourth, fifth, sixth,seventh, eighth, ninth, or tenth aspects of the invention, the sensestrand and the antisense strand of the nucleic acid sequence aretranscribed from the same nucleic acid sequence using two convergentpromoters. In another desirable embodiment, in a vector for use in anyof the above aspects of the invention, the nucleic acid sequencecomprises an inverted repeat, such that upon transcription, the nucleicacid forms a double stranded RNA.

[0038] In yet another embodiment of any of the fourth, fifth, sixth,seventh, eighth, ninth, or tenth aspects of the invention, the doublestranded RNA is at least 100, 500, 600, or 1000 nucleotides in length.In other embodiments of any of the fourth, fifth, or sixth aspects ofthe invention, the double stranded RNA is at least 10, 20, 30, 40, 50,60, 70, 80, or 90 nucleotides in length. In yet other embodiments, thenumber of nucleotides in the double stranded RNA is between 5-100nucleotides, 15-100 nucleotides, 20-95 nucleotides, 25-90 nucleotides,35-85 nucleotides, 45-80 nucleotides, 50-75 nucleotides, or 55-70nucleotides, inclusive. In still other embodiments, the number ofnucleotides in the double stranded RNA is contained in one of thefollowing ranges: 5-15 nucleotides, 15-20 nucleotides, 20-25nucleotides, 25-35 nucleotides, 35-45 nucleotides, 45-60 nucleotides,60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100nucleotides, inclusive. In other embodiments, the double stranded RNAcontains less than 50,000; 10,000; 5,000; or 2,000 nucleotides. Inaddition, the double stranded RNA may contain a sequence that is lessthan a full length RNA sequence.

[0039] In still further embodiments of any of the fourth, fifth, sixth,seventh, eighth, ninth, or tenth aspects of the invention, the cell is aplant cell or an animal cell. Desirably the animal cell is a vertebrateor mammalian cell, for example, a human cell. The cell may be ex vivo orin vivo. The cell may be a gamete or a somatic cell, for example, acancer cell, a stem cell, a cell of the immune system, a neuronal cell,a muscle cell, or an adipocyte.

[0040] In other embodiments of any of the first, second, third, seventh,eighth, ninth, or tenth aspects of the invention, the double strandedRNA is derived from a cell or a population of cells and used totransform another cell population of either the same cell type or adifferent cell type. In desirable embodiments, the transformed cellpopulation contains cells of a cell type that is related to the celltype of the cells from which the double stranded RNA was derived (e.g.,the transformation of cells of one neuronal cell type with the doublestranded RNA derived from cells of another neuronal cell type). In yetother embodiments of any of these aspects, the double stranded RNAcontains one or more contiguous or non-contiguous positions that arerandomized (e.g., by chemical or enzymatic synthesis using a mixture ofnucleotides that may be added at the randomized position). In stillother embodiments, the double stranded RNA is a randomized nucleic acidin which segments of ribonucleotides and/or deoxyribonucleotides areligated to form the double stranded RNA.

[0041] In other embodiments of any of various aspects of the invention,the double stranded RNA specifically hybridizes to a target nucleic acidbut does not substantially hybridize to non-target molecules, whichinclude other nucleic acids in the cell or biological sample having asequence that is less than 99, 95, 90, 80, or 70% identical orcomplementary to that of the target nucleic acid. Desirably, the amountof the these non-target molecules hybridized to, or associated with, thedouble stranded RNA, as measured using standard assays, is 2-fold,desirably 5-fold, more desirably 10-fold, and most desirably 50-foldlower than the amount of the target nucleic acid hybridized to, orassociated with, the double stranded RNA.

[0042] In other embodiments, the amount of a target nucleic acidhybridized to, or associated with, the double stranded RNA, as measuredusing standard assays, is 2-fold, desirably 5-fold, more desirably10-fold, and most desirably 50-fold greater than the amount of a controlnucleic acid hybridized to, or associated with, the double stranded RNA.Desirably, the double stranded RNA only hybridizes to one target nucleicacid from a cell under denaturing, high stringency hybridizationconditions. In certain embodiments, the double stranded RNA issubstantially homologous (e.g., at least 80, 90, 95, 98,or 100%homologous) to only one target nucleic acid from a cell. In otherembodiments, the double stranded RNA is homologous to multiple RNAs,such as RNAs from the same gene family. In yet other embodiments, thedouble stranded RNA is homologous to distinctly different mRNA sequencesfrom genes that are similarly regulated (e.g., developmental, chromatinremodeling, or stress response induced). In other embodiments, thedouble stranded RNA is homologous to a large number of RNA molecules,such as a double stranded RNA designed to induce a stress response orapoptosis. In other embodiments, the percent decrease in the expressionof a target nucleic acid is at least 2, 5, 10, 20, or 50 fold greaterthan the percent decrease in the expression of a non-target or controlnucleic acid. Desirably, the double stranded RNA inhibits the expressionof a target nucleic acid but has negligible, if any, effect on theexpression of other nucleic acids in the cell. Examples of controlnucleic acids include nucleic acids with a random sequence or nucleicacids known to have little, if any, affinity for the double strandedRNA.

[0043] In other embodiments of any of various aspects of the invention,at most one molecular species of double stranded RNA is inserted intoeach cell. In other embodiments, at most one vector is stably integratedinto the genome of each cell. In various embodiments, the doublestranded RNA is active in the nucleus of the transformed cell and/or isactive in the cytoplasm of the transformed cell. In various embodiments,at least 1, 10, 20, 50, 100, 500, or 1000 cells or all of the cells inthe population are selected as cells that contain or express a doublestranded RNA. In some embodiments, at least 1, 10, 20, 50, 100, 500, or1000 cells or all of the cells in the population are assayed for amodulation in the function of the cell, a modulation in the expressionof a target nucleic acid (e.g., an endogenous or pathogen gene) in thecell, and/or a modulation in the biological activity of a target protein(e.g., an endogenous or pathogen protein) in the cell.

[0044] In other embodiments, the double stranded RNA or double strandedRNA expression vector is complexed with one or more cationic lipids orcationic amphiphiles, such as the compositions disclosed in U.S. Pat.No. 4,897,355 (Eppstein et al., filed Oct. 29, 1987), U.S. Pat. No.5,264,618 (Felgner et al., filed Apr. 16, 1991) or U.S. Pat. No.5,459,127 (Felgner et al., filed Sep. 16, 1993). In other embodiments,the double stranded RNA or double stranded RNA expression vector iscomplexed with a liposomes/liposomic composition that includes acationic lipid and optionally includes another component such as aneutral lipid (see, for example, U.S. Pat. No. 5,279,833 (Rose), U.S.Pat. No. 5,283,185 (Epand), and U.S. Pat. No. 5,932,241). In yet otherembodiments, the double stranded RNA or double stranded RNA expressionvector is complexed with any other composition that is devised by one ofordinary skill in the fields of pharmaceutics and molecular biology.

[0045] Desirably, the double stranded RNA specifically hybridizes to atarget nucleic acid but does not substantially hybridize to non-targetmolecules, which include other nucleic acids in the cell or biologicalsample having a sequence that is less than 99, 95, 90, 80, or 70%identical to or complementary to that of the target nucleic acid. Inother embodiments, the percent decrease in the expression of a targetnucleic acid is at least 2, 5, 10, 20, or 50 fold greater than thepercent decrease in the expression of a non-target or control nucleicacid. Desirably, the double stranded RNA inhibits the expression of thetarget nucleic acid but has negligible, if any, effect on the expressionof other nucleic acids in the cell.

[0046] Transformation/transfection of the cell may occur through avariety of means including, but not limited to, lipofection,DEAE-dextran-mediated transfection, microinjection, protoplast fusion,calcium phosphate precipitation, viral or retroviral delivery,electroporation, or biolistic transformation. The RNA or RNA expressionvector (DNA) may be naked RNA or DNA or local anesthetic complexed RNAor DNA (Pachuk et al., supra). In yet another embodiment, the cell isnot a C. elegans cell. Desirably the vertebrate or mammalian cell hasbeen cultured for only a small number of passages (e.g., less than 30passages of a cell line that has been directly obtained from AmericanType Culture Collection), or are primary cells. In addition, desirablythe vertebrate or mammalian cell is transformed with double stranded RNAthat is not complexed with cationic lipids.

[0047] The transcription systems described herein provide advantages toother double stranded expression systems. Following transformation ofthe double stranded RNA library, cells contain hundreds to thousands ofdouble stranded RNA expression cassettes, with concomitant expression ofthat many expression cassettes. In the double stranded RNA expressionsystem of the present invention, double stranded RNA (dsRNA) expressioncassettes contained within the expression vector integrate into thechromosome of the transfected cell. Desirably, every transformed cellintegrates one of the double stranded expression cassettes. Throughexpansion of the transformed cell, episomal (non-integrated) expressionvectors are diluted out of the cell over time. Desirably notranscription occurs until the episomal expression vectors are dilutedout of the cell, such that not more than 5 episomal vectors remain inthe cell. Most desirably, no transcription occurs until all of theepisomal vectors have been diluted out of the cell, and only theintegrated expression cassette remains. The time it takes for allepisomal vectors to be removed from the cell is proportional to thereplication rate of the transformed cell, and is generally on the orderof two to several weeks of cell culture and growth. The numbers ofcopies of a dsRNA molecule in a transformed cell can be determinedusing, for example, standard PCR techniques, and thereby, the number ofepisomal vectors in a given cell can be monitored.

[0048] Once a stable integrant containing five or fewer, and desirablyno episomal expression vectors, transcription is induced, allowing dsRNAto be expressed in the cells. This method ensures that, if desired, onlyone species or not more than about five species of dsRNA is expressedper cell, as opposed to other methods that express hundreds to thousandsof double stranded species.

[0049] Another problem that can occur in other double strandedexpression systems or dsRNA delivery systems is that some dsRNAsequences, possibly in certain cell types and through certain deliverymethods, may result in an interferon response (Jaramillo et al., CancerInvest. 13:327-338, 1995). During the induction of post-transcriptionalgene silencing events, induction of an interferon response is notdesired, as this could lead to cell death and possibly to the preventionof gene silencing. An additional advantage of the present invention isthat the dsRNA delivery methods described herein are performed such thatan interferon response is inhibited or prevented.

[0050] One of the components of an interferon response is the inductionof the interferon-induced protein kinase PKR (Jaramillo et al., supra).Suppression of the interferon response and/or the PKR response, usingtechniques described herein, is desired in the cells targeted for a PTGSevent in those instances where an interferon response would otherwise beinduced. Methods for suppressing an interferon response or dsRNA stressresponse can be used in combination with any of the methods foridentifying a nucleic acid sequence that modulates the function of acell, gene expression in a cell, or the biological activity of a targetpolypeptide.

[0051] The methods of the present invention provide a means for highthroughput identification of nucleic acid sequences involved inmodulating the function of a cell, the expression of a target nucleicacid in a cell, or the biological activity of a target polypeptide in acell. By transforming a population of cells with a double stranded RNAexpression library, the effects of many PTGS events on cell function,expression of a target nucleic acid in a cell, or the biologicalactivity of a target polypeptide in a cell can be evaluatedsimultaneously, thereby allowing for rapid identification of the nucleicacid sequence involved in a cell function, target nucleic acidexpression, or biological activity of a target polypeptide of interest.

[0052] By “nucleic acid,” “nucleic acid sequence,” “double stranded RNAnucleic acid sequence,” or “double stranded RNA nucleic acid” is meant anucleic acid or a portion thereof that is free of the genes that, in thenaturally-occurring genome of the organism from which the nucleic acidsequence of the invention is derived, flank the gene. The term thereforeincludes, for example, a recombinant DNA, with or without 5′ or 3′flanking sequences that is incorporated into a vector, for example, adouble stranded RNA expression vector; into an autonomously replicatingplasmid or virus; or into the genomic DNA of a prokaryote or eukaryote;or which exists as a separate molecule (e.g., a cDNA or a genomic orcDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences.

[0053] By “double stranded RNA” is meant a nucleic acid containing aregion of two or more nucleotides that are in a double strandedconformation. In various embodiments, the double stranded RNA consistsentirely of ribonucleotides or consists of a mixture of ribonucleotidesand deoxynucleotides, such as the RNA/DNA hybrids disclosed, forexample, by WO 00/63364, filed Apr. 19, 2000 or U.S. Ser. No.60/130,377, filed Apr. 21, 1999. The double stranded RNA may be a singlemolecule with a region of self-complimentarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In various embodiments, a double stranded RNAthat consists of a single molecule consists entirely of ribonucleotidesor includes a region of ribonucleotides that is complimentary to aregion of deoxyribonucleotides. Alternatively, the double stranded RNAmay include two different strands that have a region of complimentarityto each other. In various embodiments, both strands consist entirely ofribonucleotides, one strand consists entirely of ribonucleotides and onestrand consists entirely of deoxyribonucleotides, or one or both strandscontain a mixture of ribonucleotides and deoxyribonucleotides.Desirably, the regions of complimentarily are at least 70, 80, 90, 95,98, or 100% complimentary. Desirably, the region of the double strandedRNA that is present in a double stranded conformation includes at least5, 10, 20, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides orincludes all of the nucleotides in a cDNA being represented in thedouble stranded RNA. In some embodiments, the double stranded RNA doesnot contain any single stranded regions, such as single stranded ends,or the double stranded RNA is a hairpin. Desirable RNA/DNA hybridsinclude a DNA strand or region that is an antisense strand or region(e.g., has at least 70, 80, 90, 95, 98, or 100% complimentary to atarget nucleic acid) and an RNA strand or region that is an sense strandor region (e.g., has at least 70, 80, 90, 95, 98, or 100% identity to atarget nucleic acid). In various embodiments, the RNA/DNA hybrid is madein vitro using enzymatic or chemical synthetic methods such as thosedescribed herein or those described in WO 00/63364, filed Apr. 19, 2000or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments,a DNA strand synthesized in vitro is complexed with an RNA strand madein vivo or in vitro before, after, or concurrent with the transformationof the DNA strand into the cell. In yet other embodiments, the doublestranded RNA is a single circular nucleic acid containing a sense and anantisense region, or the double stranded RNA includes a circular nucleicacid and either a second circular nucleic acid or a linear nucleic acid(see, for example, WO 00/63364, filed Apr. 19, 2000 or U.S. Ser. No.60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acidsinclude lariat structures in which the free 5′ phosphoryl group of anucleotide becomes linked to the 2′ hydroxyl group of another nucleotidein a loop back fashion.

[0054] In other embodiments, the double stranded RNA includes one ormore modified nucleotides in which the 2′ position in the sugar containsa halogen (such as flourine group) or contains an alkoxy group (such asa methoxy group) which increases the half-life of the double strandedRNA in vitro or in vivo compared to the corresponding double strandedRNA in which the corresponding 2′ position contains a hydrogen or anhydroxyl group. In yet other embodiments, the double stranded RNAincludes one or more linkages between adjacent nucleotides other than anaturally-occurring phosphodiester linkage. Examples of such linkagesinclude phosphoramide, phosphorothioate, and phosphorodithioatelinkages. In other embodiments, the double stranded RNA contains one ortwo capped strands, as disclosed, for example, by WO 00/63364, filedApr. 19, 2000 or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In otherembodiments, the double stranded RNA contains coding sequence ornon-coding sequence, for example, a regulatory sequence (e.g., atranscription factor binding site, a promoter, or a 5′ or 3′untranslated region (UTR) of an mRNA). Additionally, the double strandedRNA can be any of the at least partially double-stranded RNA moleculesdisclosed in WO 00/63364, filed Apr. 19, 2000 (see, for example, pages8-22). Any of the double stranded RNAs may be expressed in vitro or invivo using the methods described herein or standard methods, such asthose described in WO 00/63364, filed Apr. 19, 2000 (see, for example,pages 16-22).

[0055] By “double stranded RNA expression library” or “dsRNA expressionlibrary” is meant a collection of nucleic acid expression vectorscontaining nucleic acid sequences, for example, cDNA sequences orrandomized nucleic acid sequences that are capable of forming a doublestranded RNA (dsRNA) upon expression of the nucleic acid sequence.Desirably the double stranded RNA expression library contains at least10,000 unique nucleic acid sequences, more desirably at least 50,000;100,000; or 500,000 unique nucleic acid sequences, and most desirably,at least 1,000,000 unique nucleic acid sequences. By a “unique nucleicacid sequence” is meant that a nucleic acid sequence of a doublestranded RNA expression library has desirably less than 50%, moredesirably less than 25% or 20%, and most desirably less than 10% nucleicacid identity to another nucleic acid sequence of a double stranded RNAexpression library when the full length sequence are compared. Sequenceidentity is typically measured using sequence analysis software with thedefault parameters specified therein (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.

[0056] The preparation of cDNAs for the generation of double strandedRNA expression libraries is described herein. A randomized nucleic acidlibrary may also be generated as described in detail below. The doublestranded RNA expression library may contain nucleic acid sequences thatare transcribed in the nucleus or that are transcribed in the cytoplasmof the cell. A double stranded RNA expression library may be generatedusing techniques described herein.

[0057] By “target nucleic acid” is meant a nucleic acid sequence whoseexpression is modulated as a result of post-transcriptional genesilencing. As used herein, the target nucleic acid may be in the cell inwhich the PTGS event occurs or it may be in a neighboring cell, or in acell contacted with media or other extracellular fluid in which the cellthat has undergone the PTGS event is contained. Exemplary target nucleicacids include nucleic acids associated with cancer or abnormal cellgrowth, such as oncogenes, and nucleic acids associated with anautosomal dominant or recessive disorder. Desirably, the double strandedRNA inhibits the expression of an allele of a nucleic acid that has amutation associated with a dominant disorder and does not substantiallyinhibit the other allele of the nucleic acid (e.g., an allele without amutation associated with the disorder). Other exemplary target nucleicacids include host cellular nucleic acids or pathogen nucleic acidsrequired for the infection or propagation of a pathogen, such as avirus, bacteria, yeast, protozoa, or parasite.

[0058] By “target polypeptide” is meant a polypeptide whose biologicalactivity is modulated as a result of post-transcriptional genesilencing. As used herein, the target polypeptide may be in the cell inwhich the PTGS event occurs or it may be in a neighboring cell, or in acell contacted with media or other extracellular fluid in which the cellthat has undergone the PTGS event is contained.

[0059] As used herein, by “randomized nucleic acids” is meant nucleicacids, for example, those that are at least 100, 500, 600, or 1000nucleotides in length, constructed from RNA isolated from a particularcell type. In other embodiments, the nucleic acids are at least 10, 20,30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In yet otherembodiments, the number of nucleotides in the nucleic acids is between5-100 nucleotides, 15-100 nucleotides, 20-95 nucleotides, 25-90nucleotides, 35-85 nucleotides, 45-80 nucleotides, 50-75 nucleotides, or55-70 nucleotides, inclusive. In still other embodiments, the number ofnucleotides in the nucleic acids is contained in one of the followingranges: 5-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-35nucleotides, 35-45 nucleotides, 45-60 nucleotides, 60-70 nucleotides,70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides, inclusive.In other embodiments, the nucleic acids contain less than 50,000;10,000; 5,000; or 2,000 nucleotides. A randomized nucleic acid librarymay be constructed in a number of ways. For example, it may beconstructed from existing cDNA libraries. In one example, the EDNAlibraries are shuffled using the “Gene Shuffling” technology of MaxygenCorp. The cDNA sequences are amplified using inefficient PCR either byrestricting elongation time or through the use of manganese. A libraryof recombinants is created, and the library is finally amplified by PCRand cloned into vectors. In a second method, existing cDNA libraries aredigested with an endonuclease to generate fragments of 10 to 300 basepairs. Alternatively, the cDNA libraries are digested to generateshorter fragments of, for example, 5 to 50 base pairs, 5 to 40 basepairs, 5 to 20 base pairs, 5 to 10 base pairs, or 10 to 20 base pairs,inclusive. If the fragments are to contain 5′ OH and 3′ PO₄ groups, theyare dephosphorylated using alkaline phosphatase and phosphorylated usingpolynucleotide kinase. These dsDNA fragments are then ligated to formlarger molecules, and are size selected. In a third example, randomizednucleic acid libraries are created by using random priming of EDNAlibraries (using random hexamers and Klenow) to generate short fragmentsof 20 to 100 nucleotides. Alternatively, shorter fragments are generatedthat contain, for example, 5 to 50 nucleotides, 5 to 40 nucleotides, 5to 20 nucleotides, 5 to 10 nucleotides, or 10 to 20 nucleotides,inclusive. These fragments are then ligated randomly to give a desiredsized larger fragment.

[0060] Alternatively, a randomized nucleic acid library can be generatedfrom random sequences of oligonucleotides. For example, DNA or RNAoligonucleotides may be prepared chemically. Random DNA sequences mayalso be prepared enzymatically using terminal transferase in thepresence of all dNTPs. Random RNA molecules may be prepared using NDPsand NDP phosphorylase. The random sequences may be 10 to 300 bases inlength. Alternatively, shorter random sequences are used that contain,for example, 5 to 50 bases, 5 to 40 bases, 5 to 20 bases, 5 to 10 bases,or 10 to 20 bases, inclusive. The sequences are ligated to form thedesired larger sequence using RNA ligase. Alternatively these sequencesmay be ligated chemically. The oligonucleotides are phosphorylated atthe 5′ position using polynucleotide kinase or by chemical methods,prior to ligation enzymatically. Chemical ligations can utilize a 5′ PO₄and a 3′ OH group or a5′ OH and a 3′ PO₄ group.

[0061] Alternatively, a randomized nucleic acid library can be generatedby converting the random DNA sequences into dsDNA sequences using DNApolymerase (Klenow), dNTP and random heteromeric primers, and the RNAsequences are converted into dsDNA sequences by reverse transcriptaseand Klenow. After converting into DNA (ss or ds) the sequences are thenamplified by PCR. The dsDNA fragments can also be ligated to give largerfragments of a desired size.

[0062] The randomized nucleic acids may be cloned into a vector, forexample, an expression vector, as a double stranded RNA transcriptioncassette. The sequence of the nucleic acid may not be known at the timethe vector is generated. The randomized nucleic acid may contain codingsequence or non-coding sequence, for example, a regulatory sequence(e.g., a transcription factor binding site, a promoter, or a 5′ or 3′untranslated region (UTR)of an mRNA).

[0063] By “Cre-mediated double recombination” is meant two nucleic acidrecombination events involving loxP sites that are mediated by Crerecombinase. A Cre-mediated double recombination event can occur, forexample, as illustrated in FIG. 1.

[0064] By “function of a cell” is meant any cell activity that can bemeasured or assessed. Examples of cell function include, but are notlimited to, cell motility, apoptosis, cell growth, cell invasion,vascularization, cell cycle events, cell differentiation, celldedifferentiation, neuronal cell regeneration, and the ability of a cellto support viral replication. The function of a cell may also be toaffect the function, gene expression, or the polypeptide biologicalactivity of another cell, for example, a neighboring cell, a cell thatis contacted with the cell, or a cell that is contacted with media orother extracellular fluid that the cell is contained in.

[0065] By “apoptosis” is meant a cell death pathway wherein a dying celldisplays a set of well-characterized biochemical hallmarks that includecytolemmal membrane blebbing, cell soma shrinkage, chromatincondensation, nuclear disintegration, and DNA laddering. There are manywell-known assays for determining the apoptotic state of a cell,including, and not limited to: reduction of MTT tetrazolium dye, TUNELstaining, Annexin V staining, propidium iodide staining, DNA laddering,PARP cleavage, caspase activation, and assessment of cellular andnuclear morphology. Any of these or other known assays may be used inthe methods of the invention to determine whether a cell is undergoingapoptosis.

[0066] By “polypeptide biological activity” is meant the ability of atarget polypeptide to modulate cell function. The level of polypeptidebiological activity may be directly measured using standard assays knownin the art. For example, the relative level of polypeptide biologicalactivity may be assessed by measuring the level of the mRNA that encodesthe target polypeptide (e.g., by reverse transcription-polymerase chainreaction (RT-PCR) amplification or Northern blot analysis); the level oftarget polypeptide (e.g., by ELISA or Western blot analysis); theactivity of a reporter gene under the transcriptional regulation of atarget polypeptide transcriptional regulatory region (e.g., by reportergene assay, as described below); the specific interaction of a targetpolypeptide with another molecule, for example, a polypeptide that isactivated by the target polypeptide or that inhibits the targetpolypeptide activity (e.g., by the two-hybrid assay); or thephosphorylation or glycosylation state of the target polypeptide. Acompound, such as a dsRNA, that increases the level of the targetpolypeptide, mRNA encoding the target polypeptide, or reporter geneactivity within a cell, a cell extract, or other experimental sample isa compound that stimulates or increases the biological activity of atarget polypeptide. A compound, such as a dsRNA, that decreases thelevel of the target polypeptide, mRNA encoding the target polypeptide,or reporter gene activity within a cell, a cell extract, or otherexperimental sample is a compound that decreases the biological activityof a target polypeptide.

[0067] By “assaying” is meant analyzing the effect of a treatment, be itchemical or physical, administered to whole animals, cells, tissues, ormolecules derived therefrom. The material being analyzed may be ananimal, a cell, a tissue, a lysate or extract derived from a cell, or amolecule derived from a cell. The analysis may be, for example, for thepurpose of detecting altered cell function, altered gene expression,altered endogenous RNA stability, altered polypeptide stability, alteredpolypeptide levels, or altered polypeptide biological activity. Themeans for analyzing may include, for example, antibody labeling,immunoprecipitation, phosphorylation assays, glycosylation assays, andmethods known to those skilled in the art for detecting nucleic acids.In some embodiments, assaying is conducted under selective conditions.

[0068] By “modulates” is meant changing, either by a decrease or anincrease. As used herein, desirably a nucleic acid sequence decreasesthe function of a cell, the expression of a target nucleic acid in acell, or the biological activity of a target polypeptide in a cell byleast 20%, more desirably by at least 30%, 40%, 50%, 60% or 75%, andmost desirably by at least 90%. Also as used herein, desirably a nucleicacid sequence increases the function of a cell, the expression of atarget nucleic acid in a cell, or the biological activity of a targetpolypeptide in a cell by at least 1.5-fold to 2-fold, more desirably byat least 3-fold, and most desirably by at least 5-fold.

[0069] By “a decrease” is meant a lowering in the level of: a) protein(e.g., as measured by ELISA or Western blot analysis); b) reporter geneactivity (e.g., as measured by reporter gene assay, for example,β-galactosidase, green fluorescent protein, or luciferase activity); c)mRNA (e.g., as measured by RT-PCR or Northern blot analysis relative toan internal control, such as a “housekeeping” gene product, for example,β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)); or d) cellfunction, for example, as assayed by the number of apoptotic, mobile,growing, cell cycle arrested, invasive, differentiated, ordedifferentiated cells in a test sample. In all cases, the lowering isdesirably by at least 20%, more desirably by at least 30%, 40%, 50%,60%, 75%, and most desirably by at least 90%. As used herein, a decreasemay be the direct or indirect result of PTGS.

[0070] By “an increase” is meant a rise in the level of: a) protein(e.g., as measured by ELISA or Western blot analysis); b) reporter geneactivity (e.g., as measured by reporter gene assay, for example,β-galactosidase, green fluorescent protein, or luciferase activity); c)mRNA (e.g., as measured by RT-PCR or Northern blot analysis relative toan internal control, such as a “housekeeping” gene product, for example,β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)); or d) cellfunction, for example, as assayed by the number of apoptotic, mobile,growing, cell cycle arrested, invasive, differentiated, ordedifferentiated cells in a test sample. Desirably, the increase is byat least 1.5-fold to 2-fold, more desirably by at least 3-fold, and mostdesirably by at least 5-fold. As used herein, an increase may be theindirect result of PTGS. For example, the double stranded RNA mayinhibit the expression of a protein, such as a suppressor protein, thatwould otherwise inhibit the expression of another nucleic acid.

[0071] By “alteration in the level of gene expression” is meant a changein transcription, translation, or mRNA or protein stability such thatthe overall amount of a product of the gene, i.e., mRNA or polypeptide,is increased or decreased.

[0072] By “reporter gene” is meant any gene that encodes a product whoseexpression is detectable and/or able to be quantitated by immunological,chemical, biochemical, or biological assays. A reporter gene productmay, for example, have one of the following attributes, withoutrestriction: fluorescence (e.g., green fluorescent protein), enzymaticactivity (e.g., β-galactosidase, luciferase, chloramphenicolacetyltransferase), toxicity (e.g., ricin A), or an ability to bespecifically bound by an additional molecule (e.g., an unlabeledantibody, followed by a labelled secondary antibody, or biotin, or adetectably labelled antibody). It is understood that any engineeredvariants of reporter genes that are readily available to one skilled inthe art, are also included, without restriction, in the foregoingdefinition.

[0073] By “protein” or “polypeptide” or “polypeptide fragment” is meantany chain of more than two amino acids, regardless of post-translationalmodification (e.g., glycosylation or phosphorylation), constituting allor part of a naturally-occurring polypeptide or peptide, or constitutinga non-naturally occurring polypeptide or peptide.

[0074] By “promoter” is meant a minimal sequence sufficient to directtranscription of a gene. Also included in this definition are thosetranscription control elements (e.g., enhancers) that are sufficient torender promoter-dependent gene expression controllable in a celltype-specific, tissue-specific, or temporal-specific manner, or that areinducible by external signals or agents; such elements, which arewell-known to skilled artisans, may be found in a 5′ or 3′ region of agene or within an intron. Desirably a promoter is operably linked to anucleic acid sequence, for example, a cDNA or a gene in such a way as topermit expression of the nucleic acid sequence.

[0075] By “operably linked” is meant that a gene and one or moretranscriptional regulatory sequences, e.g., a promoter or enhancer, areconnected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequences.

[0076] By “expression vector” is meant a DNA construct that contains atleast one promoter operably linked to a downstream gene or coding region(e.g., a cDNA or genomic DNA fragment that encodes a protein,optionally, operatively linked to sequence lying outside a codingregion, an antisense RNA coding region, or RNA sequences lying outside acoding region). Transfection or transformation of the expression vectorinto a recipient cell allows the cell to express RNA encoded by theexpression vector. An expression vector may be a genetically engineeredplasmid, virus, or artificial chromosome derived from, for example, abacteriophage, adenovirus, retrovirus, poxvirus, or herpesvirus.

[0077] By “transformation” or “transfection” is meant any method forintroducing foreign molecules into a cell (e.g., a bacterial, yeast,fungal, algal, plant, insect, or animal cell, particularly a vertebrateor mammalian cell). The cell may be in an animal. Lipofection,DEAE-dextran-mediated transfection, microinjection, protoplast fusion,calcium phosphate precipitation, viral or retroviral delivery,electroporation, and biolistic transformation are just a few of thetransformation/transfection methods known to those skilled in the art.The RNA or RNA expression vector (DNA) may be naked RNA or DNA or localanesthetic complexed RNA or DNA (Pachuk et al., supra). Other standardtransformation/transfection methods and other RNA and/or DNA deliveryagents (e.g., a cationic lipid, liposome, or bupivacaine) are describedin WO 00/63364, filed Apr. 19, 2000 (see, for example, pages 18-26).Commercially available kits can also be used to deliver RNA or DNA to acell. For example, the Transmessenger Kit from Qiagen, an RNA kit fromXeragon Inc., and an RNA kit from DNA Engine Inc. (Seattle, Wash.) canbe used to introduce single or double stranded RNA into a cell.

[0078] By “transformed cell” or “transfected cell” is meant a cell (or adescendent of a cell) into which a nucleic acid molecule, for example, adouble stranded RNA or double stranded expression vector has beenintroduced, by means of recombinant nucleic acid techniques. Such cellsmay be either stably or transiently transfected.

[0079] By “selective conditions” is meant conditions under which aspecific cell or group of cells can be selected for. For example, theparameters of a fluorescence-activated cell sorter (FACS) can bemodulated to identify a specific cell or group of cells. Cell panning, atechnique known to those skilled in the art, is another method thatemploys selective conditions.

[0080] As use herein, by “optimized” is meant that a nucleic acidfragment is generated through inefficient first strand synthesis (e.g.,reverse transcription (RT) and/or RT/second strand synthesis (RT-SSS)using Klenow or other enzymes and/or RT-PCR or PCR, to be of aparticular length. Desirably the length of the nucleic acid fragment isless than a fall length cDNA or is 100, 500, 600, or 1000 nucleotides inlength. In other embodiments, the nucleic acid fragment is at least 10,20, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In yet otherembodiments, the number of nucleotides in the nucleic acid fragment isbetween 5-100 nucleotides, 15-100 nucleotides, 20-95 nucleotides, 25-90nucleotides, 35-85 nucleotides, 45-80 nucleotides, 50-75 nucleotides, or55-70 nucleotides, inclusive. In still other embodiments, the number ofnucleotides in the nucleic acid fragment is contained in one of thefollowing ranges: 5-15 nucleotides, 15-20 nucleotides, 20-25nucleotides, 25-35 nucleotides, 35-45 nucleotides, 45-60 nucleotides,60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100nucleotides, inclusive. In other embodiments, the nucleic acid fragmentcontains less than 50,000; 10,000; 5,000; or 2,000 nucleotides.Optimization of the length of a nucleic acid can be achieved duringfirst strand or second strand synthesis of a desired nucleic acid bylowering Mg⁺⁺ concentrations to no less than the nucleotideconcentrations; by adding Mn⁺⁺to the reaction to achieve the desiredsize selection (e.g., by replacing Mg⁺⁺ completely, or by adding Mn⁺⁺ atvarying concentrations along with Mg⁺⁺); by decreasing and/or limitingconcentrations of dNTP(s) to effect the desired fragment size; by usingvarious concentrations of ddNTP(s) along with standard or optimalconcentrations of dNTP(s), to achieve varying ratios, to obtain thedesired fragment size; by using limited and controlled exonucleasedigestion of the fragment following RT, RT-SSS, RT-PCR, or PCR; or by acombination of any of these methods.

[0081] As used herein, by “sized selected” is meant that a nucleic acidof a particular size is selected for use in the construction of dsRNAexpression libraries as described herein. Desirably the size selectednucleic acid is less than a full length cDNA sequence or at least 100,500, 600, or 1000 nucleotides in length. In other embodiments, thenucleic acid is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90nucleotides in length. In yet other embodiments, the number ofnucleotides in the nucleic acid is between 5-100 nucleotides, 15-100nucleotides, 20-95 nucleotides, 25-90 nucleotides, 35-85 nucleotides,45-80 nucleotides, 50-75 nucleotides, or 55-70 nucleotides, inclusive.In still other embodiments, the number of nucleotides in the nucleicacid is contained in one of the following ranges: 5-15 nucleotides,15-20 nucleotides, 20-25 nucleotides, 25-35 nucleotides, 35-45nucleotides, 45-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides,80-90 nucleotides, or 90-100 nucleotides, inclusive. In otherembodiments, the nucleic acid contains less than 50,000; 10,000; 5,000;or 2,000 nucleotides. For example, a nucleic acid may be size selectedusing size exclusion chromatography (e.g., as size exclusion Sepharosematrices) according to standard procedures (see, for example, Sambrook,Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.),Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0082] By “under conditions that inhibit or prevent an interferonresponse or a dsRNA stress response” is meant conditions that prevent orinhibit one or more interferon responses or cellular RNA stressresponses involving cell toxicity, cell death, an anti-proliferativeresponse, or a decreased ability of a dsRNA to carry out a PTGS event.These responses include, but are not limited to, interferon induction(both Type 1 and Type II), induction of one or more interferonstimulated genes, PKR activation, 2′5′-OAS activation, and anydownstream cellular and/or organismal sequelae that result from theactivation/induction of one or more of these responses. By “organismalsequelae” is meant any effect(s) in a whole animal, organ, or morelocally (e.g., at a site of injection) caused by the stress response.Exemplary manifestations include elevated cytokine production, localinflammation, and necrosis. Desirably the conditions that inhibit theseresponses are such that not more than 95%, 90%, 80%, 75%, 60%, 40%, or25%, and most desirably not more than 10% of the cells undergo celltoxicity, cell death, or a decreased ability to carry out a PTGS event,compared to a cell not exposed to such interferon response inhibitingconditions, all other conditions being equal (e.g., same cell type, sametransformation with the same dsRNA expression library.

[0083] Apoptosis, interferon induction, 2′5′ OAS activation/induction,PKR induction/activation, anti-proliferative responses, and cytopathiceffects are all indicators for the RNA stress response pathway.Exemplary assays that can be used to measure the induction of an RNAstress response as described herein include a TUNEL assay to detectapoptotic cells, ELISA assays to detect the induction of alpha, beta andgamma interferon, ribosomal RNA fragmentation analysis to detectactivation of 2′5′OAS, measurement of phosphorylated eIF2a as anindicator of PKR (protein kinase RNA inducible) activation,proliferation assays to detect changes in cellular proliferation, andmicroscopic analysis of cells to identify cellular cytopathic effects(see, e.g., Example 11). Desirably, the level of an interferon responseor a dsRNA stress response in a cell transformed with a double strandedRNA or a double straned RNA expression vector is less than 20, 10, 5,or2-fold greater than the corresponding level in a mock-transfectedcontrol cell under the same conditions, as measured using one of theassays described herein. In other embodiments, the level of aninterferon response or a dsRNA stress response in a cell transformedwith a double stranded RNA or a double straned RNA expression vectorusing the methods of the present invention is less than 500%, 200%,100%, 50%, 25%, or 10% greater than the corresponding level in acorresponding transformed cell that is not exposed to such interferonresponse inhibiting conditions, all other conditions being equal.Desirably, the double straned RNA does not induce a global inhibition ofcellular transcription or translation.

[0084] By “specifically hybridizes” is meant a double stranded RNA thathybridizes to a target nucleic acid but does not substantially hybridizeto other nucleic acids in a sample (e.g., a sample from a cell) thatnaturally includes the target nucleic acid, when assayed underdenaturing conditions. In one embodiment, the amount of a target nucleicacidhybridized to, or associated with, the double stranded RNA, asmeasured using standard assays, is 2-fold, desirably 5-fold, moredesirably 10-fold, and most desirably 50-fold greater than the amount ofa control nucleic acid hybridized to, or associated with, the doublestranded RNA.

[0085] By “high stringency conditions” is meant hybridization in 2×SSCat 40° C. with a DNA probe length of at least 40 nucleotides. For otherdefinitions of high stringency conditions, see F. Ausubel et al.,Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley &Sons, New York, N.Y., 1994, hereby incorporated by reference.

[0086] Conditions and techniques that can be used to prevent aninterferon response or dsRNA stress response during the screeningmethods of the present invention are described herein.

BRIEF DESCRIPTION OF THE DRAWING

[0087]FIG. 1 is a schematic representation of a strategy to isolateclonally pure stable integrants that contain a single expression unitisolated from cells transfected with a double-stranded RNA encoding acDNA library.

[0088]FIG. 2 is a schematic illustration of the production of effectorRNAs in cells expressing PSA. The PSA expression cassette used to createthe transient PSA expression cell line is depicted at the top of thefigure. Expression of PSA is driven by the HCMV IE promoter and the SV40polyadenylation signal (pA). Only sequences 3′ of the PSA initiationcodon have been used in these vectors. The effector RNA expressioncassettes are shown below the PSA expression cassette and are designedto express PSA sense RNA, PSA antisense RNA, and PSA dsRNA. Expressionof the effector RNAs is under the control of the T7 promoter (T7p).Transcription from T7p is catalyzed by T7 RNA polymerase, which issupplied by co-transfecting a T7 RNA polymerase expression plasmid (notshown). Control effector RNA cassettes expressing irrelevant RNAsderived from the Herpes simplex virus glycoprotein D gene were includedas controls. The 600 base pair sequence from the Herpes simplex gD geneis from Herpes Simplex virus 2 strain 12 and maps to the coding regiondownstream of the gD initiation codon.

[0089]FIG. 3 is a bar graph illustrating silencing of PSA expression bydsRNA. PSA levels in the supernates of transfected cells were determinedby ELISA and are plotted as percent expression of the PSA untreatedcontrol. The PSA untreated control shown at the left is normalized to100%. PSA levels in the supernates of cells transfected with the variouseffector PSA or control RNAs are shown by the shaded and open barsrespectively. All data shown is from day two post-transfection. Datafrom later time points were similar to the day two time point.

[0090]FIG. 4 is a schematic illustration of the RNA stress responsepathway, also known as the Type 1 interferon response.

DETAILED DESCRIPTION OF THE INVENTION

[0091] Post-transcriptional gene silencing (PTGS) can be used as a toolto identify and validate specific unknown genes involved in cellfunction, gene expression, and polypeptide biological activity. Althoughthe use of PTGS as a validation strategy is well documented ininvertebrates and plants, its use in identification of genes thatmodulate cell function, gene expression, or polypeptide biologicalactivity, as described below, is novel. Since novel genes are likely tobe identified through the methods of the present invention, PTGS isdeveloped for use in validation and to identify novel targets for use intherapies for diseases, for example, cancer, neurological disorders,obesity, leukemia, lymphomas, and other disorders of the blood or immunesystem.

[0092] The present invention features methods to identify unknowntargets that result in the modulation of a particular phenotype, analteration of gene expression in a cell, or an alteration in polypeptidebiological activity in a cell, using either a library based screeningapproach or a non-library based approach to identify nucleic acids thatinduce gene silencing. The present invention also allows thedetermination of function of a given sequence. These methods involve thedirect delivery of in vitro transcribed double stranded RNA (dsRNA), aswell as plasmid-based systems that direct the cell to make its owndsRNA. To avoid problems associated with transfection efficiency,plasmids are designed to contain a selectable marker to ensure thesurvival of only those cells that have taken up plasmid DNA. One groupof plasmids directs the synthesis of dsRNA that is transcribed in thecytoplasm, while another group directs the synthesis of dsRNA that istranscribed in the nucleus.

Identification of Genes by Assaying for a Modulation in Cell Function

[0093] Functional identification of novel genes can be accomplishedthrough the use of a number of different assays. For example, cells maybe assayed for cell motility, apoptosis, cell growth, cell invasion,vascularization, cell cycle events, cell differentiation, celldedifferentiation, neuronal cell regeneration, or the ability to supportviral replication, as well as other cell functions known in the art.Methods for carrying out such functional assays are well known and aredescribed, for example, in Platet and Garcia (Invasion Metastasis18:198-208, 1998-1999); Harper et al. (Neuroscience 88:257-267, 1999);and Tomaselli et al. (J. Cell Biol. 105:2347-2358, 1987), and are alsodescribed below.

[0094] Functional identification of nucleic acid sequences involved inmodulating a particular cell function may be carried out by comparingcells transfected with a dsRNA to control cells that have not beentransformed with a dsRNA or that have been mock-transfected, in afunctional assay. A cell that has taken up sequences unrelated to aparticular function will perform in the particular assay in a mannersimilar to the control cell. A cell experiencing PTGS of a gene involvedin the particular function will exhibit an altered ability to perform inthe functional assay compared to the control.

[0095] The percent modulation of a particular cell function thatidentifies a nucleic acid sequence that modulates the function of a cellwill vary depending on the assay, phenotype, and the particular nucleicacid affected by PTGS. For each assay, the percent modulation canreadily be determined by one skilled in the art, when used inconjunction with controls, as described herein. Desirably the modulationis at least 20%, more desirably at least 30%, 40%, 50%, 60%, 75%, andmost desirably at least 90% compared to the control. An increase in thefunction of a cell can also be measured in terms of fold increase, wheredesirably, the increase is at least 1.5-fold to 5-fold compared to thecontrol.

[0096] Alternatively, the function of a cell may be to affect thefunction, gene expression, or polypeptide biological activity of anothercell, for example, a neighboring cell, a cell that is contacted with thecell in which a PTGS event occurs, or a cell that is contacted withmedia or other extracellular fluid that the cell in which a PTGS eventoccurs is contained in. For example, a cell experiencing PTGS of a genemay modulate cell motility, apoptosis, cell growth, cell invasion,vascularization, cell cycle events, cell differentiation, celldedifferentiation, neuronal cell regeneration, or the ability to supportviral replication of a nearby cell, or a cell that is exposed to mediaor other extracellular fluid in which the transfected cell in which aPTGS event occurs was once contained. This can be tested by removing themedia in which a cell experiencing a PTGS event is occurring and placingit on a separate cell or population of cells. If the function of theseparate cell or population of cells is modulated, compared to a cell orpopulation of cells receiving media obtained from cells that had beenmock transfected, then one or more of the cells experiencing a PTGSevent can affect the function of another cell. The identity of thenucleic acid sequence that causes the modulation can be identified withrepeated rounds of selection.

[0097] In another method, a single cell experiencing a PTGS event can beplaced in proximity of a cell or a population of cells that was nottransfected with dsRNA, and the effect of this placement is evaluatedfor a modulation in the function of the cell or population of cells. Ifthe function of the non-transfected cell or population of cells ismodulated, compared to a cell or population of cells in proximity of acell that was mock transfected, then the cell experiencing a PTGS eventcontains a nucleic acid sequence that can affect the function of anothercell. This nucleic acid sequence can be identified using techniquesdescribed herein. Identification of Genes Using Differential GeneExpression

[0098] Differential gene expression analysis can be used to identify anucleic acid sequence that modulates the expression of a target nucleicacid in a cell. Alterations in gene expression induced by gene silencingcan be monitored in a cell into which a dsRNA has been introduced. Forexample, differential gene expression can be assayed by comparingnucleic acids expressed in cells into which dsRNA has been introduced tonucleic acids expressed in control cells that were not transfected withdsRNA or that were mock-transfected. Gene array technology can be usedin order to simultaneously examine the expression levels of manydifferent nucleic acids. Examples of methods for such expressionanalysis are described by Marrack et al. (Current Opinions in Immunologyb 12:206-209, 2000); Harkin (Oncologist 5:501-507, 2000); Pelizzari etal. (Nucleic Acids Res. 28:4577-4581, 2000); and Marx (Science289:1670-1672, 2000).

Identification of Genes by Assaying Polypeptide Biological Activity

[0099] Novel nucleic acid sequences that modulate the biologicalactivity of a target polypeptide can also be identified by examiningpolypeptide biological activity. Various polypeptide biologicalactivities can be evaluated to identify novel genes according to themethods of the invention. For example, the expression of a targetpolypeptide(s) may be examined. Alternatively, the interaction between atarget polypeptide(s) and another molecule(s), for example, anotherpolypeptide or a nucleic acid may be assayed. Phosphorylation orglycosylation of a target polypeptide(s) may also be assessed, usingstandard methods known to those skilled in the art.

[0100] Identification of nucleic acid sequences involved in modulatingthe biological activity of a target polypeptide may be carried out bycomparing the polypeptide biological activity of a cell transfected witha dsRNA to a control cell that has not been transfected with a dsRNA orthat has been mock-transfected. A cell that has taken up sequencesunrelated to a particular polypeptide biological activity will performin the particular assay in a manner similar to the control cell. A cellexperiencing PTGS of a gene involved in the particular polypeptidebiological activity will exhibit an altered ability to perform in thebiological assay, compared to the control.

Insertion of Single Units into the Chromosome and Generation of a CellLine Containing a Single dsRNA Expression Library Integrant

[0101] The present invention involves the generation of a target cellline in which the dsRNA expression library is subsequently introduced.Through the use of site-specific recombination, single integrants ofdsRNA expression cassettes are generated at the same locus of all cellsin the target cell line, allowing uniform expression of the dsRNA in allof the integrants. A dsRNA expression library derived from various celllines is used to create a representative library of stably integratedcells, each cell within the target cell line containing a singleintegrant. Cre/lox, Lambda-Cro repressor, and Flp recombinase systems orretroviruses are used to generate these singular integrants of dsRNAexpression cassettes in the target cell line (Satoh et al., J. Virol.74:10631-10638, 2000; Trinh et al., J. Immunol. Methods 244:185-193,2000; Serov et al., An. Acad. Bras. Cienc. 72:389-398, 2000; Grez et al,Stem Cells. 16:235-243, 1998; Habu et al., Nucleic Acids Symp. Ser.2:295-296, 1999; Haren et al., Annu. Rev. Microbiol. 53:245-281, 1999;Baer et al., Biochemistry 39:7041-7049, 2000; Follenzi et al., Nat.Genet. 25:217-222, 2000; Hindmarsh et al., Microbiol. Mol. Biol. Rev.63:836-843, 1999; Darquet et al., Gene Ther. 6:209-218, 1999; Darquet etal., Gene Ther. 6:209-218, 1999; Yu et al, Gene 223:77-81, 1998; Darquetet al., Gene Ther. 4:1341-1349, 1997; and Koch et al., Gene 249:135-144,2000). These systems are used singularly to generate singular insertionclones, and also in combination.

[0102] The following exemplary sequence specific integrative systems useshort target sequences that allow targeted recombination to be achievedusing specific proteins: FLP recombinase, bacteriophage Lambdaintegrase, HIV integrase, and pilin recombinase of Salmonella (Seng etal. Construction of a Flp “exchange cassette” contained vector and genetargeting in mouse ES cell] A book chapter PUBMED entry 11797223-ShengWu Gong Cheng Xue Bao. 2001 September;17(5):566-9., Liu et al., NatGenet. 2001 January 1;30(1):66-72., Awatramani et al., Nat Genet. 2001November;29(3):257-9., Heidmann and Lehner, Dev Genes Evol. 2001September;211(8-9):458-65, Schaft et al.,Genesis. 2001 September;31(1):6-10, Van Duyne, Annu Rev Biophys Biomol Struct. 2001;30:87-104.,Lorbach et al., J Mol Biol. 2000 March 10;296(5):1175-81., Darquet etal., Gene Ther. 1999 February;6(2):209-18., Bushman and Miller, J Virol.1997 January;71(1):458-64., Fulks et al.,J Bacteriol. 1990January;172(1):310-6). A singular integrant is produced by randomlyinserting the specific sequence (e.g., loxP in the cre recombinasesystem) and selecting or identifying the cell that contains a singularintegrant that supports maximal expression. For example, integrants thatshow maximal expression following random integration can be identifiedthrough the use of reporter gene sequences associated with theintegrated sequence. The cell can be used to specifically insert theexpression cassette into the site that contains the target sequenceusing the specific recombinase, and possibly also remove the expressioncassette that was originally placed to identify the maximally expressingchromosomal location. A skilled artisan can also produce singularintegrants using retroviral vectors, which integrate randomly andsingularly into the eukaryotic genome. In particular, singularintegrants can be produced by inserting retroviral vectors that havebeen engineered to contain the desired expression cassette into a naivecell and selecting for the chromosomal location that results in maximalexpression (Michael et al., EMBO Journal, vol 20: pages 2224-2235, 2001;Reik and Murrell., Nature, vol. 405, page 408-409, 2000; Berger et al.,Molecular Cell, vol. 8, pages 263-268). One may also produce a singularintegrant by cotransfecting the bacterial RecA protein with or withoutnuclear localization signal along with sequences that are homologous tothe target sequence (e.g., a target endogenous sequence or integratedtransgene sequence). Alternatively, a nucleic acid sequence that encodesa RecA protein with nuclear localization signals can be cotransfected(Shibata et al., Proc Natl Acad Sci U S A. 2001 July 17;98(15):8425-32.Review., Muyrers et al., Trends Biochem Sci. 2001 May;26(5):325-31.,Paul et al., Mutat Res. 2001 June 5;486(1): 11-9., Shcherbakova et al.,Mutat Res. 2000 February 16;459(1):65-71., Lantsov. Mol Biol (Mosk).1994 May-June;28(3):485-95).

[0103] An example utilizing such methods is detailed below.

Creation of the Target Cell Line

[0104] Target cell lines are the same cell lines as the ones from whichthe dsRNA expression libraries will be derived. Target cells are createdby transfecting the selected cell line with a bicistronic plasmidexpressing a selectable marker, such as G418 and the reporter gene GFP.The plasmid also bears a loxP site. Plasmids integrate randomly into thechromosome through the process of illegitimate recombination at afrequency of 10⁻⁴. Following transfection, cells containing integrantsare selected by culturing the cells in the presence of G418 at aconcentration determined earlier in a kill curve analysis. About a dozenG418-resistant colonies are expanded and relative GFP expression levelsare determined using flow cytometry. DNA from the cells is analyzed bySouthern blot analysis to determine integrant copy number. Severalsingle copy integrants exhibiting the highest GFP expression levels arethen selected as the target cell lines. GFP expression is monitoredbecause dsRNA encoding templates are then integrated into the locicontaining the loxP, GFP, and G418 cassettes in a site-specific fashion,and it is important to ensure that these loci are transcriptionallyactive. Since cells are selected on the basis of G418 resistance and GFPexpression, integration of the plasmid DNA can occur at the loxP site,destroying its function. Several cell lines are therefore chosen toreasonably ensure that at least one integrant has an intact loxP site.

Double Stranded RNA Expression Library Construction and Site-specificRecombination into the Target Cell Line

[0105] A cDNA library or a randomized library is constructed from RNAisolated from selected cell lines. cDNAs or randomized nucleic acids inthe size range of at least 100 to 1000 nucleotides, for example, 500 to600 nucleotides are optimized during synthesis or are size-selectedprior to cloning. In other embodiments, the nucleic acids are at least10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length. In yetother embodiments, the number of nucleotides in the nucleic acids isbetween 5-100 nucleotides, 15-100 nucleotides, 20-95 nucleotides, 25-90nucleotides, 35-85 nucleotides, 45-80 nucleotides, 50-75 nucleotides, or55-70 nucleotides, inclusive. In still other embodiments, the number ofnucleotides in the nucleic acids is contained in one of the followingranges: 5-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-35nucleotides, 35-45 nucleotides, 45-60 nucleotides, 60-70 nucleotides,70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides, inclusive.In other embodiments, the nucleic acid contains less than 50,000;10,000; 5,000; or 2,000 nucleotides. Each cDNA or randomized nucleicacid is then cloned into a plasmid vector as a dsRNA transcriptioncassette flanked by two convergent promoters (such as T7 promoters asdescribed herein). The promoters are transcriptionally regulated suchthat they are off until induced, for example, using a tet ON/OFF system(Forster et al., Nucleic Acids Res. 27:7708-710, 1999; Liu et al.,Biotechniques 24:624-628, 6,30-632, 1998; and Gatz, Methods Cell Biol.50:411-424, 1995). The plasmid also contains the hygromycin resistancegene and an inverted loxP site. The cDNA plasmid library or randomizedplasmid library is then co-transfected into the target cell line with aplasmid expressing Cre recombinase, which catalyzes site-specificrecombination of the transfected cDNA plasmid or randomized nucleic acidplasmid at the inverted loxP site into the chromosomal locus containingthe GFP gene and loxP site (see FIG. 1). The use of the Cre/lox systemallows the efficient integration of a plasmid into the chromosome (everytransfected cell is predicted to undergo a plasmid integration event).Other site-specific recombination strategies can also be utilized. Thisresults in having every integration to occur at the same site, therebyobviating potential problems with loci dependent expression.

[0106] Two days following transfection, cells are incubated in thepresence of hygromycin to kill untransfected cells and to select forstable integrants. Transcription of dsRNA is induced, and selected cellsare assayed for an alteration in cell function, the biological activityof a target polypeptide, or differential gene expression. Cellsexpressing dsRNA corresponding to a target nucleic acid exhibit analtered function, for example, increased or decreased cell invasion,motility, apoptosis, growth, differentiation, dedifferentiation, orregeneration, or the ability of the cell to support viral replication.Cells exhibiting altered function are then expanded and the sequence ofthe integrant is determined. Targets are identified and validated usingdsRNA specific for the identified target, or other non-PTGS mediatedmethods, for example antisense technology.

[0107] The regulated transcription system of the present inventionprovides an advantage to other double stranded expression systems.Following transfection of the dsRNA library, cells contain hundreds tothousands of dsRNA expression cassettes, with concomitant expression ofthat many expression cassettes. In the dsRNA expression system of thepresent invention, dsRNA expression cassettes contained within theexpression vector integrate into the chromosome of the transfected cell.As described in detail below, every transfected cell integrates one ofthe double stranded expression cassettes. Desirably no transcriptionoccurs until the episomal (non-integrated) expression vectors arediluted out of the cell such that not more than 5 episomal vectorsremain in the cell. Most desirably no transcription occurs until all ofthe episomal (non-integrated) expression vectors are diluted out of thecell and only the integrated expression cassette remains (a processusually taking about two to several weeks of cell culture). At this timetranscription is induced, allowing dsRNA to be expressed in the cells.This method ensures that only one species of dsRNA is expressed percell, as opposed to other methods that express hundreds to thousands ofdouble stranded species. The use of the above-described system resultsin the loss of all but one expression cassette, which in turn, permitsthe rapid screening of libraries without requiring screening multiplepools of libraries to identify the target gene.

Non-library Approaches for the Identification of a Nucleic Acid Sequencethat Modulates Cell Function, Gene Expression in a Cell, or theBiological Activity of a Target Polypeptide in a Cell Through PTGS

[0108] Nucleic acid sequences that modulate cell function, geneexpression in a cell, or the biological activity of a target polypeptidein a cell may also be identified using non-library based approachesinvolving PTGS. For example, a single known nucleic acid sequenceencoding a polypeptide with unknown function or a single nucleic acidfragment of unknown sequence and/or function can be made into a doublestranded RNA molecule. This dsRNA is then transfected into a desiredcell type and the cell is assayed for modulations in cell function, geneexpression of a target nucleic acid in the cell, or the biologicalactivity of a target polypeptide in the cell, using methods describedherein. A modulation in cell function, gene expression in the cell, orthe biological activity of a target polypeptide in the cell identifiesthe nucleic acid of the dsRNA as a nucleic acid the modulates thespecific cell function, gene expression, or the biological activity of atarget polypeptide. As a single dsRNA species is transfected into thecells, the nucleic acid sequence responsible for the modulation isreadily identified. This non-library based approach to nucleic acididentification is desirably used under conditions that inhibit aninterferon response or dsRNA stress response. Such conditions aredescribed in detail herein.

[0109] The discovery of novel genes through the methods of the presentinvention may lead to the generation of novel therapeutics. For example,genes that decrease cell invasion may be used as targets for drugdevelopment, such as for the development of cytostatic therapeutics foruse in the treatment of cancer. Development of such therapeutics isimportant because currently available cytotoxic anticancer agents arealso toxic for normal rapidly dividing cells. In contrast, a cytostaticagent may only need to check metastatic processes, and by inference,slow cell growth, in order to stabilize the disease. In another example,genes that increase neuronal regeneration may be used to developtherapeutics for the treatment, prevention, or control of a number ofneurological diseases, including Alzheimer's disease and Parkinson'sdisease. Genes that are involved in the ability to support viralreplication and be used as targets in anti-viral therapies. Suchtherapies may be used to treat, prevent, or control viral diseasesinvolving human immunodeficiency virus (HIV), hepatitis C virus (HCV),hepatitis B virus (HBV), and human papillomavirus (HPV). The efficaciesof therapeutics targeting the genes identified according to the presentinvention can be further tested in cell culture assays, as well as inanimal models.

The use of Vertebrate or Mammalian Cell Lines for Identification ofNucleic Acid Sequences that Modulate Cell Function, Expression of aTarget Nucleic Acid, or Biological Activity of a Target Polypeptide

[0110] While the use of the present invention is not limited tovertebrate or mammalian cells, such cells can be used to carry out thenucleic acid identification methods described herein. Desirably thevertebrate or mammalian cells used to carry out the present inventionare cells that have been cultured for only a small number of passages(e.g., less than 30 passages of a cell line that has been obtaineddirectly from American Type Culture Collection), or are primary cells.In addition, vertebrate or mammalian cells can be used to carry out thepresent invention when the dsRNA being transfected into the cell is notcomplexed with cationic lipids.

[0111] The following examples are to illustrate the invention. They arenot meant to limit the invention in any way. For example, it is notedthat any of the following examples can be used with double stranded RNAsof any length. The methods of the present invention can be readilyadapted by one skilled in the art to utilize double stranded RNAs of anydesired length.

EXAMPLE 1 Design and Delivery of Vectors for Intracellular Synthesis ofdsRNA for Library Based Screening Approaches to Nucleic AcidIdentification Using PTGS

[0112] PTGS is induced when dsRNA is made intracellularly. The librarybased screening approaches to nucleic acid identification through PTGSmay require that dsRNA reside in certain cellular compartments in orderto exert its effect. Therefore, expression plasmids that transcribedsRNA in the cytoplasm and in the nucleus are utilized. There are twoclasses of nuclear transcription vectors: one that is designed toexpress polyadenylated dsRNA (for example, a vector containing an RNApolymerase II promoter and a poly A site) and one that expressesnon-adenylated dsRNA (for example, a vector containing an RNA polymeraseII promoter and no poly A site, or a vector containing a T7 promoter).Different cellular distributions are predicted for the two species ofRNA; both vectors are transcribed in the nucleus, but the ultimatedestinations of the RNA species are different intracellular locations.Intracellular transcription may also utilize bacteriophage T7 and SP6RNA polymerase, which may be designed to transcribe in the cytoplasm orin the nucleus. Alternatively, Qbeta replicase RNA-dependent RNApolymerase may be used to amplify dsRNA. Viral RNA polymerases, eitherDNA and RNA dependent, may also be used. Alternatively, dsRNAreplicating polymerases can be used. Cellular polymerases such as RNAPolymerase I, II, or III or mitochondrial RNA polymerase may also beutilized. Both the cytoplasmic and nuclear transcription vectors containan antibiotic resistance gene to enable selection of cells that havetaken up the plasmid. Cloning strategies employ chain reaction cloning(CRC), a one-step method for directional ligation of multiple fragments(Pachuk et al., Gene 243:19-25, 2000). Briefly, the ligations utilizebridge oligonucleotides to align the DNA fragments in a particular orderand ligation is catalyzed by a heat-stable DNA ligase, such asAmpligase, available from Epicentre.

Inducible or Repressible Transcription Vectors for the Generation of adsRNA Expression Library

[0113] If desired, inducible and repressible transcription systems canbe used to control the timing of the synthesis of dsRNA. Inducible andrepressible regulatory systems involve the use of promoter elements thatcontain sequences that bind prokaryotic or eukaryotic transcriptionfactors upstream of the sequence encoding dsRNA. In addition, thesefactors also carry protein domains that transactivate or transrepressthe RNA polymerase II. The regulatory system also has the ability tobind a small molecule (e.g., a coinducer or a corepressor). The bindingof the small molecule to the regulatory protein molecule (e.g., atranscription factor) results in either increased or decreased affinityfor the sequence element. Both inducible and repressible systems can bedeveloped using any of the inducer/transcription factor combinations bypositioning the binding site appropriately with respect to the promotersequence. Examples of previously described inducible/repressible systemsinclude lac, ara, Steroid-RU486, and ecdysone—Rheogene, Lac (Cronin etal. Genes & Development 15: 1506-1517, 2001), ara (Khlebnikov et al., JBacteriol. 2000 December; 182(24):7029-34) , ecdysone (Rheogene,www.rheogene.com), RU48 (steroid, Wang X J, Liefer K M, Tsai S, O'MalleyB W, Roop D R., Proc Natl Acad Sci U S A. 1999 July 20;96(15):8483-8),tet promoter (Rendal et al., Hum Gene Ther. 2002 January;13(2):335-42.and Lamartina et al., Hum Gene Ther. 2002 January;13(2):199-210), or apromoter disclosed in WO 00/63364, filed Apr. 19, 2000.

Nuclear Transcription Vectors for the Generation of a Nuclear dsRNAExpression Library

[0114] Nuclear transcription vectors for use in library based screeningapproaches to identify nucleic acids that modulate cell function, geneexpression, or the biological activity of a target polypeptide aredesigned such that the target sequence is flanked on one end by an RNApolymerase II promoter (for example, the HCMV-IE promoter) and on theother end by a different RNA polymerase II promoter (for example, theSCMV promoter). Other promoters that can be used include other RNApolymerase II promoters, an RNA polymerase I promoter, an RNA polymeraseIII promoter, a mitochondrial RNA polymerase promoter, or a T7 or SP6promoter in the presence of T7 or SP6 RNA polymerase, respectively,containing a nuclear localization signal. Bacteriophage or viralpromoters may also be used. The promoters are regulatedtranscriptionally (for example, using a tet ON/OFF system (Forster etal., supra; Liu et al., supra; and Gatz, supra) such that they are onlyactive in either the presence of a transcription-inducing agent or uponthe removal of a repressor. A single chromosomal integrant is selectedfor, and transcription is induced in the cell to produce the nucleardsRNA.

[0115] Those vectors containing a promoter recognized by RNA Pol I, RNAPol II, or a viral promoter in conjunction with co-expressed proteinsthat recognize the viral promoter, may also contain optional sequenceslocated between each promoter and the inserted cDNA. These sequences aretranscribed and are designed to prevent the possible translation of atranscribed cDNA. For example, the transcribed RNA is synthesized tocontain a stable stem-loop structure at the 5′ end to impede ribosomescanning. Alternatively, the exact sequence is irrelevant as long as thelength of the sequence is sufficient to be detrimental to translationinitiation (e.g., the sequence is 200 nucleotides or longer). The RNAsequences can optionally have sequences that allow polyA addition,intronic sequences, an HIV REV binding sequence, Mason-Pfizer monkeyvirus constitutive transport element(CTE) (U.S. Pat. No. 5,880,276,filed Apr. 25, 1996), and/or self splicing intronic sequences.

[0116] To generate dsRNA, two promoters can be placed on either side ofthe target sequence, such that the direction of transcription from eachpromoter is opposing each other. Alternatively, two plasmids can becotransfected. One of the plasmids is designed to transcribe one strandof the target sequence while the other is designed to transcribe theother strand. Single promoter constructs may be developed such that twounits of the target sequence are transcribed in tandem, such that thesecond unit is in the reverse orientation with respect to the other.Alternate strategies include the use of filler sequences between thetandem target sequences.

Cytoplasmic Transcription Vectors for the Generation of a CytoplasmicdsRNA Expression Library

[0117] Cytoplasmic transcription vectors for use in library basedscreening approaches to identifying nucleic acids that modulate cellfunction, gene expression, or the biological activity of a targetpolypeptide in a cell using PTGS are made according to the followingmethod. This approach involves the transcription of a single strandedRNA template (derived from a library) in the nucleus, which is thentransported into the cytoplasm where it serves as a template for thetranscription of dsRNA molecules. The DNA encoding the ssRNA isintegrated at a single site in the target cell line as described for thenuclear RNA expression library, thereby ensuring the synthesis of onlyone species of dsRNA in a cell, each cell expressing a different dsRNAspecies.

[0118] A desirable approach is to use endogenous polymerases such as themitochondrial polymerase in animal cells or mitochondrial andchloroplast polymerases in plant cells for cytoplasmic and mitochondrial(e.g., chloroplast) expression to make dsRNA in the cytoplasm. Thesevectors are formed by designing expression constructs that containmitochondrial or chloroplast promoters upstream of the target sequence.As described above for nuclear transcription vectors, dsRNA can begenerated using two such promoters placed on either side of the targetsequence, such that the direction of transcription from each promoter isopposing each other. Alternatively, two plasmids can be cotransfected.One of the plasmids is designed to transcribe one strand of the targetsequence while the other is designed to transcribe the other strand.Single promoter constructs may be developed such that two units of thetarget sequence are transcribed in tandem, such that the second unit isin the reverse orientation with respect to the other. Alternatestrategies include the use of filler sequences between the tandem targetsequences.

[0119] Alternatively, cytoplasmic expression of dsRNA for use in librarybased screening approaches is achieved by a single subgenomic promoteropposite in orientation with respect to the nuclear promoter. Thenuclear promoter generates one RNA strand that is transported into thecytoplasm, and the singular subgenomic promoter at the 3′ end of thetranscript is sufficient to generate its antisense copy by an RNAdependent RNA polymerase to result in a cytoplasmic dsRNA species.

Target Cell Line Development for use with Cytoplasmic dsRNA ExpressionLibraries

[0120] The target cell line, using the vector containing the G418cassette, GFP, and loxP site is designed as described above.

Development of a Cytoplasmic dsRNA Expression Library

[0121] Double stranded RNA expression libraries are generated byinserting cDNA or randomized sequences (as described herein) into anexpression vector containing a single nuclear promoter (RNA polymeraseI, RNA polymerase II, or RNA polymerase III), which allows transcriptionof the insert sequence. It is desirable that this nuclear promoteractivity is regulated transcriptionally (for example, using a tet ON/OFFsystem described, for example, by Forster et al., supra; Liu et al.,supra; and Gatz, supra), such that the promoters are only active ineither the presence of a transcription-inducing agent or upon theremoval of a repressor. This ensures that transcription is not induceduntil episomal copies of the vector(s) are diluted out. Vectors alsocontain a selectable marker, such as the hygromycin resistance gene, anda loxP site. The expression vectors are integrated into the target cellline by methods previously described in this application using Crerecombinase (other site-specific recombinative strategies can beemployed, as described previously).

[0122] At two days post-transfection, cells are subjected to hygromycinselection using concentrations established in kill curve assays.Surviving cells are cultured in hygromycin to select for cells bearingintegrated vectors and to dilute out episomal copies of the vector(s).At this point transcription is induced, and a single stranded RNA(ssRNA) species derived from the insert sequence is transcribed in thenucleus from the nuclear promoter in the inserted vector. The insert isdesigned such that the insert sequences in the transcript are flanked bybi-directional promoters of RNA bacteriophages (for example, Qbeta orMS2, RNA dependent RNA polymerase promoters) or cytoplasmic viralRNA-dependent RNA polymerase promoter sequences (for example, those ofSindbis or VEEV subgenomic promoters). The nuclear transcript istranslocated to the cytoplasm where it acts as a template for dsRNA byan RNA dependent RNA polymerase, which may be provided throughco-transfection of a vector that encodes an RNA-dependent RNApolymerase. Alternatively, an integrated copy of the polymerase may beused.

EXAMPLE 2 Generation of Templates for In Vitro Transcription of dsRNAfor Non-library Based Approaches for Identification of Nucleic AcidsUsing PTGS

[0123] Nucleic acid sequences that modulate cell function, geneexpression in a cell, or the biological activity of a target polypeptidein a cell may also be identified using non-library based approachesinvolving PTGS. A single known nucleic acid sequence encoding apolypeptide with unknown function or a single nucleic acid fragment ofunknown sequence and/or function can be made into a double stranded RNAmolecule. This dsRNA is then transfected into a desired cell type andassayed for modulations in cell function, gene expression in the cell,or the biological activity of a target polypeptide in the cell, usingmethods described herein. A modulation in cell function, gene expressionin the cell, or the biological activity of a target polypeptide in thecell identifies the nucleic acid of the dsRNA as a nucleic acid themodulates the specific cell function, gene expression, or the biologicalactivity of a target polypeptide. This non-library based approach tonucleic acid identification is desirably used under conditions thatinhibit an interferon response or dsRNA stress response. Such conditionsare described in detail below.

[0124] Nucleic acid fragments generated, for example, by PCR orrestriction endonuclease digestion, encoding the respective targetsequences were used as templates for in vitro transcription reactions.PCR fragments are superior to plasmid templates for the synthesis ofdiscrete sized RNA molecules. The PCR fragments encoded at least 20-50or 100 to 1000, for example, 500 to 600 nucleotides (nts) of the targetsequence and were derived from the target mRNA. Known target sequenceswere obtained from GenBank and or other DNA sequence databases. Targetsequences were also obtained from cellular RNAs that were generated intocDNAs to create a number of different dsRNA molecules. Accordingly, itis possible that the sequence and/or function of the target sequence wasnot known at the time the dsRNA was generated.

[0125] Templates for sense target RNAs were generated by placing thebacteriophage T7 promoter at the 5′ end of the target coding strandwhile antisense RNA templates contained the T7 promoter at the 5′ end ofthe non-coding strand. This was achieved by encoding the T7 promoter atthe 5′ ends of the respective PCR primers. Alternatively SP6 promoters,or a combination of SP6 and T7 promoters may be used.

[0126] PCR was performed by conventional methods. The use of both PCRtemplates in equimolar amounts in an in vitro transcription reactionresulted in primarily dsRNA. The use of two separate fragments has beenfound to be superior to the use of one PCR fragment containing two T7promoters, one located at each end of the target sequence, presumablydue to transcription interference that occurs during transcription ofthe dual promoter template. Following PCR amplification, the DNA wassubjected to Proteinase K digestion and phenol-chloroform extraction toremove contaminating RNases. Following ethanol precipitation, the DNAwas resuspended in RNase-free water at a concentration of 1 to 3 μg/μl.

[0127] As an alternative to phenol-chloroform extraction, DNA can bepurified in the absence of phenol using standard methods such as thosedescribed by Li et al. (WO 00/44914, filed Jan. 28, 2000).Alternatively, DNA that is extracted with phenol and/or chloroform canbe purified to reduce or eliminate the amount of phenol and/orchloroform. For example, standard column chromatography can be used topurify the DNA (WO 00/44914, filed Jan. 28, 2000).

EXAMPLE 3 In vitro RNA Transcription and RNA Analysis

[0128] In vitro transcription reactions are carried out using theRiboprobe Kit (Promega Corp.), according to the manufacturer'sdirections. The template DNA is as described above. Following synthesis,the RNA is treated with RQ1 DNase (Promega Corp.) to remove templateDNA. The RNA is then treated with Proteinase K and extracted withphenol-chloroform to remove contaminating RNases. The RNA is ethanolprecipitated, washed with 70% ethanol, and resuspended in RNase-freewater. Aliquots of RNA are removed for analysis and the RNA solution isflash frozen by incubating in an ethanol-dry ice bath. The RNA is storedat −80° C.

[0129] As an alternative to phenol-chloroform extraction, RNA can bepurified in the absence of phenol using standard methods such as thosedescribed by Li et al. (WO 00/44914, filed Jan. 28, 2000).Alternatively, RNA that is extracted with phenol and/or chloroform canbe purified to reduce or eliminate the amount of phenol and/orchloroform. For example, standard column chromatography can be used topurify the RNA (WO 00/44914, filed Jan. 28, 2000). dsRNA is made bycombining equimolar amounts of PCR fragments encoding antisense RNA andsense RNA, as described above, in the transcription reaction. Singlestranded antisense or sense RNA is made by using a single species of PCRfragment in the reaction. The RNA concentration is determined byspectrophotometric analysis, and RNA quality is assessed by denaturinggel electrophoresis and by digestion with RNase T1, which degradessingle stranded RNA.

[0130] An mRNA library is produced using Qbeta bacteriophage, byligating the mRNAs to the flank sequences that are required for Qbetareplicase function (Qbeta flank or Qbeta flank plus P1), using RNAligase. The ligated RNAs are then transformed into bacteria that expressQbeta replicase and the coat protein. Single plaques are then inoculatedinto fresh bacteria. All plaques are expected to carry transgenesequences. Each plaque is grown in larger quantities in bacteria thatproduce the Qbeta polymerase, and RNA is isolated from the bacteriophageparticles. Alternatively, if the Qbeta flank plus P1 is used to generatethe library (e.g., P1=MS2, VEEV, or Sindbis promoter sequences), thesevectors can be used to carry out the in vitro transcription along withthe cognate polymerase. The in vitro made dsRNA is then used totransfect cells.

RNA Delivery

[0131] In vitro made dsRNA is directly added to the cell culture mediumat concentrations ranging from 50 μg/ml to 500 μg/ml. Uptake of dsRNA isalso facilitated by electroporation using those conditions required forDNA uptake by the desired cell type. RNA uptake is also mediated bylipofection using any of a variety of commercially available andproprietary cationic lipids, DEAE-dextran-mediated transfection,microinjection, protoplast fusion, calcium phosphate precipitation,viral or retroviral delivery, or biolistic transformation. The RNA isnaked RNA or a local anesthetic RNA complex. Modulation of cellfunction, gene expression, or polypeptide biological activity is thenassessed in the transfected cells.

[0132] Some dsRNA sequences, possibly in certain cell types and throughcertain delivery methods, may result in an interferon response. Duringthe screening methods of the present invention, induction of aninterferon response is not desired, as this would lead to cell death andpossibly to the prevention of gene silencing.

[0133] One of the components of an interferon response is the inductionof the interferon-induced protein kinase PKR. Suppression of theinterferon response and/or the PKR response is desired in the targetcells. The dsRNA delivery methods described herein are performed suchthat an interferon response or dsRNA stress response is not included. Itis recognized, however, that certain conditions might present with aninduction of the interferon response. To prevent such a response, anumber of other strategies may be employed with any of the abovedescribed screening methods to identify a nucleic acid that modulatescell function, gene expression, or the polypeptide biological activityof a cell, as described herein.

[0134] To prevent an interferon response, interferon and PKR responsesare silenced in the target cells using a dsRNA species directed againstthe mRNAs that encode proteins involved in the response. Desirablyinterferon response promoters are silenced using dsRNA. Alternatively,the expression of proteins that bind the interferon response element isabolished using dsRNA techniques.

[0135] In an alternative strategy, interferon and PKR knockout celllines are created through approaches utilizing expression cassettes thatencode an antisense RNA and ribozymes directed to the cellular mRNAsthat encode the proteins involved in the response. Knockout cells arecreated by standard gene knockout technologies using homologousrecombination to alter target sequences, using homologous DNA alone, oras complexes of RecA protein and single stranded DNA homologous to thetarget sequence(s). Interferon response element (IRE) sequences,sequences that encode transcription factors that bind IRE sequences, thepromoter and/or gene sequences that encode proteins in the PKR andinterferon response pathways are molecules that are targeted forknockout.

[0136] If desired, proteins involved in gene silencing such as Dicer orArgonaut can be overexpressed or activated to increase the amount ofinhibition of gene expression (Beach et al., WO 01/68836, filed Mar. 16,2001).

EXAMPLE 4 Cytoplasmic Transcription Vectors for Non-library BasedApproaches to Nucleic Acid Identification Using PTGS

[0137] Double stranded RNA molecules for use in non-library basedmethods for the identification of nucleic acids that modulate cellfunction, gene expression of a target nuclec acid, or target polypeptidebiological activity in a cell can also be generated through the use ofcytoplasmic transcription vectors. Such vectors are generated as nowdescribed.

[0138] The PCR fragments generated for in vitro transcription templates,as described above, are inserted into a cloning vector containing one T7promoter located just outside the polylinker region. Such a vector ispZERO blunt (Promega Corp.). The PCR fragment is cloned into arestriction site in the polylinker in such a way that the fragment's T7promoter is distal to the vector's promoter. The resulting vectorcontains the target sequence flanked by two T7 promoters; transcriptionfrom this vector occurs in converging directions. Convergenttranscription is desired for these intracellular vectors, due to theuncertainty of getting sense and antisense vectors into the same cell inhigh enough and roughly equivalent amounts. In addition, the localconcentration of antisense and sense RNAs with respect to each other ishigh enough to enable dsRNA formation when the dual promoter constructis used.

[0139] A hygromycin resistance cassette is cloned into the pZERO bluntvector as well. The hygromycin resistance cassette contains thehygromycin resistance gene under the control of the Herpes Simplex Virus(HSV) thymidine kinase promoter and the SV40 polyadenlyation signal. Thecassette is in a plasmid vector and is flanked at both ends by apolylinker region enabling ease of removal and subsequent cloning.Hygromycin selection was chosen because of the rapidity of death inducedby hygromycin as well as extensive in-house experience with hygromycinselection. Alternatively, other selection methods known to those skilledin the art may be used.

[0140] The vectors are transfected into the desired cells using standardtransformation or transfection techniques described herein, and thecells are assayed for the ability of the dsRNA molecules encoded by thevectors to modulate cell function, gene expression of a target nuclecacid, or the biological activity of a target polypeptide, as describedherein.

EXAMPLE 5 Analysis of RNA From Transfected Cells

[0141] Regardless of whether a library based screening approach or anon-library based approach was used to identify nucleic acid sequences,in order to measure the level of dsRNA effector molecule within thecell, as well as the amount of target mRNA within the cell, a two-stepreverse transcription PCR reaction was performed with the ABI PRISM™7700 Sequence Detection System. Total RNA was extracted from cellstransfected with dsRNA or a plasmid from a dsRNA expression libraryusing Trizol and DNase. Two to three different cDNA synthesis reactionswere performed per sample; one for human GAPDH (a housekeeping gene thatshould be unaffected by the effector dsRNA), one for the target mRNA,and/or one for the sense strand of the expected dsRNA molecule (effectormolecule). Prior to cDNA synthesis of dsRNA sense strands, the RNAsample was treated with T1 RNase. The cDNA reactions were performed inseparate tubes using 200 ng of total RNA and primers specific for therelevant RNAs. The cDNA products of these reactions were used astemplates for subsequent PCR reactions to amplify GAPDH, the targetcDNA, and/or the sense strand copied from the dsRNA. All RNA wasquantified relative to the internal control, GAPDH.

EXAMPLE 6 Target Sequence Identification

[0142] To identify the target sequence affected by a dsRNA, using any ofthe above-described methods, DNA is extracted from expanded cell lines(or from the transfected cells if using a non-integrating dsRNA system)according to methods well known to the skilled artisan. The dsRNAencoding sequence of each integrant (or non-integrated dsRNA molecule ifusing a non-library based method) is amplified by PCR using primerscontaining the sequence mapping to the top strand of the T7 promoter (orany other promoter used to express the dsRNA). Amplified DNA is thencloned into a cloning vector, such as pZERO blunt (Promega Corp.), andthen sequenced. Sequences are compared to sequences in GenBank and/orother DNA databases to look for sequence identity or homology usingstandard computer programs. If the target mRNA remains unknown, the mRNAis cloned from the target cell line using primers derived from thecloned dsRNA by established techniques (Sambrook et al., supra). Targetvalidation is then carried out as described herein.

[0143] In the stably integrated dsRNA expression system described above,despite efforts to reduce negative position effects, inefficient dsRNAsynthesis by PCR methods may occur. This can be circumvented by rescuingthe integrated cDNA or randomized nucleic sequences into replicatingplasmids. Rescued plasmids are amenable to amplification in bacteria andto sequencing. Rescue is achieved by re-transfecting the population ofcells transfected with the dsRNA expression library with the rescueplasmid and a plasmid encoding Cre recombinase. The rescue plasmidcarries a bacterial origin of replication, a bacterial antibioticselection marker, an SV40 origin of replication, and an SV40 T antigenexpression cassette, as well as loxP sites positioned as an invertedrepeat to allow Cre-mediated double recombination. The SV40-based originof replication in the rescue plasmid allows amplification of rescuedsequences in the integrated cells. Following rescue, higher levels oftranscription are anticipated, thereby favoring dsRNA formation. Thecells are then screened for modulations in cell function, target nucleicacid expression, or target polypeptide biological activity changes asdescribed herein.

EXAMPLE 7 Prevention of an Interferon Response During Gene Silencing

[0144] As discussed above, during the above-described screening methods,induction of an interferon response is not desired, as this would leadto cell death, anti-proliferative responses, and possibly to preventionof gene silencing. One of the components of an interferon response isthe induction of the interferon-induced protein kinase PKR. Suppressionof the interferon response and/or the PKR response is desired in thetarget cells. The dsRNA delivery methods described herein are performedsuch that an interferon response is not included. It is recognized,however, that certain conditions might present with an induction of theinterferon response. To prevent such a response, a number of otherstrategies may be employed with any of the above described screeningmethods to identify a nucleic acid that modulates cell function, geneexpression, or the polypeptide biological activity of a cell, asdescribed herein.

[0145] To prevent an interferon response in a system involving stableintegration of the nucleic acid containing the dsRNA expressioncassette, the vectors used to generate either the loxP integrant or thevector that encodes the dsRNA expression cassette are designed tocontain sequences that encode proteins that block the PKR response, suchas the Vaccinia virus protein E3 (Romano et al., Molecular and CellularBiology 18:7304-7316, 1998; Accession No. M36339), or a cellular proteinP58^(IPK), which the influenza virus mobilizes to block PKR (Gale etal., Microbiology and Molecular Biology Reviews 64:239-280, 2000;Accession No. XM_(—)032882). Several other viral proteins have also beenidentified (e.g., Hepatitis C E2; Accession No. S72725) and may besimilarly used. These proteins can be expressed in the desired celltypes or in animals through the use of any of a number of commerciallyavailable mammalian expression vectors or vertebrate expression vectors.Such vectors can be obtained from a number of different manufacturersincluding Invitrogen (Carlsbad, Calif.) Promega ((Madison, Wis.), orClontech (Palo Alto, Calif.). An example of such a vector is the pCI-neoMammalian Expression Vector from Promega.

[0146] Regardless of whether nucleic acid encoding a dsRNA is stablyintegrated into a chromosome or is not integrated into a chromosome, thefollowing methods may be used to prevent an interference response in anyof the screening methods of the present invention. In one example of aninterferon avoidance strategy, interferon and PKR responses are silencedin the target cells using a dsRNA species directed against the mRNAsthat encode proteins involved in the response. Desirably interferonresponse promoters are silenced using dsRNA. Alternatively, theexpression of proteins that bind the interferon response element isabolished using dsRNA techniques.

[0147] In an alternative strategy, interferon and PKR knockout celllines are created through approaches utilizing expression cassettes thatencode an antisense RNA and ribozymes directed to the cellular mRNAsthat encode the proteins involved in the response. Knockout cells arecreated by standard gene knockout technologies using homologousrecombination to alter target sequences, using homologous DNA alone, oras complexes of RecA protein and single stranded DNA homologous to thetarget sequence(s). Interferon response element (IRE) sequences,sequences that encode transcription factors that bind IRE sequences, thepromoter and/or gene sequences that encode proteins in the PKR andinterferon response pathways are molecules that are targeted forknockout.

[0148] In yet another alternative, chimeric oligonucleotides may be usedto alter target sequences. Methods for inhibiting expression ofpolypeptides through chimeric oligonucleotides are well known in the art(Igoucheva and Yoon, Human Gene Therapy 11:2307-2312, 2000).

EXAMPLE 8 Functional Screening for Cell Invasion

[0149] Cell invasion is one cell function that may be evaluated in thesearch for novel genes that are modulated using the methods describedherein. Matrigel, a biological extracellular matrix, has propertiessimilar to that of a reconstituted basement membrane and has been usedto measure the invasive potential of tumor cells (Platet and Garcia,supra). Cells transfected with randomized or cDNA libraries that havebeen cloned into PTGS vectors are monitored for their capacity to invadematrigel invasion chambers. Cells that have taken up sequences unrelatedto invasion invade the matrigel as efficiently as vector-transfectedcontrol cells. Cells experiencing PTGS of genes that are involved incell invasion invade much less efficiently. If the dsRNA expressioncassette is stably integrated in a chromosome, these cells are retrievedand second and third rounds of selection are carried out in order toisolate specific nucleic acid sequences relevant to cell invasion. Theeffect of these sequences on invasion is ultimately confirmed by theirability to block the formation of tumors in animal models.

[0150] Several human cell lines, for example, MDA-MB-231, used by Platetand Garcia (supra), SKBr3, and MCF-7ADR, a more metastatic variant ofMCF-7. MDA-MB-231 breast cancer cells (obtained from the American TypeCulture Collection) are also transfected with cDNA libraries orrandomized nucleic acid libraries constructed into the vectors describedabove. Desirably all cells in this assay contain a single copy of atransfected gene, as described above.

[0151] Cells cultured in commercially available 24- or 96-well formattedsystems are used to carry out the cell invasion assay. As this screeningprotocol relies upon repeated rounds of selection, it may be desirableto keep the cell numbers in each well low enough that enrichment is seenin each succeeding round, yet high enough to recover sufficient cells toculture within a reasonable time period. Therefore, culture conditionsthat result in invasion by greater than 50% of the cells and that stillpermit recovery from the surface of the matrigel are made optimal.Non-invasive (NIH3T3 cells) or poorly invasive (MCF7) cell lines areanalyzed in parallel as negative controls for invasion.

[0152] Initially, triplicate cultures of half-log order dilutions from10² to 10⁶ cells per well are plated. Cells are then recovered by“scrubbing” with a sterile cotton swab in fresh culture media and areseeded into 96-well plates. The number of invasive cells in the matrigelis quantified using either an MTT-based assay (Sasaki and Passaniti,Biotechniques 24:1038-1043, 1998) or a fluorescent indicator (Gohla etal., Clin. Exp. Metastasis 14:451-458, 1996).

[0153] Once the appropriate cell densities for the assay have beenempirically determined, stable transfected cells are plated in thematrigel cell invasion chambers. Each experiment includes the followingcontrols: a sample of untransfected cells as a reference culture;untransfected cells treated with anti-invasive chemotherapeutic agents,such as taxol or doxorubicin, as a positive control for inhibition ofinvasion; cells transfected with empty vectors to confirm that thevector alone had no effects on invasion; and cells transfected cellswith genes that are known to block invasion in this assay, such asestrogen receptors or TIMP-2 (Kohn et al., Cancer Research 55:1856-1862,1995; and Woodhouse et al., Cancer (Supplement) 80:1529-1536, 1997).

[0154] Cells that fail to invade the matrigel are removed from each wellto the corresponding wells of a 96-well plate and cultured untilmacroscopic colonies are visible. It is important to collect cells atmore than one time point after plating, since the time it takes for PTGSto be effective may vary, and it may be that different genes are activeat different times after plating. Once the cells are transferred to96-well plates, they are diluted out and taken through successive roundsof re-screening in the invasion assay in order to expand and isolatecell lines with altered invasive ability. As the population becomes moreand more enriched for cells with a non-invasive phenotype, the reductionin invasive cells in the matrigel can be better quantified via MTT orfluorescence assays. Ultimately, a large panel of cloned double-stablecell lines is generated.

[0155] This assay can also be carried out with cells into which a dsRNAis not stably integrated into a chromosome. The assay is conductedessentially as described above except that multiple rounds of selectionand re-screening are not necessary since the cell is transfected withonly one dsRNA species. Thus, the target(s) of the PTGS event is readilyidentifiable using the cloning and sequencing techniques describedabove.

EXAMPLE 9 Downregulation of HIV Using HIV-derived dsRNA and Inhibitorsof the Interferon Response Pathway

[0156] During the course of HIV infection, the viral genome is reversetranscribed into a DNA template that is integrated into the hostchromosome of infected dividing cells. The integrated copy is now ablueprint from which more HIV particles are made. Several cell linesthat contain integrated copies of a defective HIV genome, HIVgpt (strainHXB2) have been created. The HIVgpt genome contains a deletion of theHIV envelope gene; all other HIV proteins are encoded. The plasmid usedto create these cell lines, HIVgpt, was obtained from the AIDS Researchand Reference Reagent Program Catalog. Stably integrated cell lines weremade with human rhabdomyosarcoma (RD) cells. The lines were made bytransfecting cells with the plasmid followed by selection of cells inmycophenolic acid. The HIVgpt genome encodes a mycophenolic acid (MPA)resistance gene in place of the envelope gene and thereby confersresistance to MPA. Cells resistant to MPA were clonally amplified. Themedia from the cultured clonally expanded cells was assayed for thepresence of p24 (an HIV gag polypeptide that is secretedextracellularly). All cell lines were positive for p24, as assessedusing a p24 ELISA assay kit (Coulter, Fullerton, Calif.). The cell linesalso make non-infectious particles which can be rescued into infectiousparticles by co-expression of an HIV envelope protein.

[0157] The HIVgpt cell lines are used as a model system with which todownregulate HIV expression via PTGS. Plasmids encoding a 600 nt senseRNA, a 600 nt antisense RNA, or a 600 bp double stranded RNA (dsRNA),mapping to the same coordinates of the gag gene of HIV strain HXB2 areused to transfect cells (the map from which the coordinates are based isfound at GenBank Accession number K03455, HIV(HXB2), complete genome,and the gag RNAs used in this study map to coordinates 901-1500).Expression of the RNAs is from T7 RNA polymerase promoter(s) located atthe 5′ end of the gag sense strand, at the 5′ end of the antisensestrand, or at converging positions at the 5′ ends of both the sense andanti-sense strands, respectively. These encoded RNAs are not designed tobe able to make protein (i.e., they do not have a cap, a poly A tail, orthe native initiation codon). Transcription of the RNAs is catalyzed byT7 RNA polymerase, provided from a second co-transfected T7 RNApolymerase expression plasmid. Control plasmids expressing a similarsized sense RNA, antisense RNA, and dsRNA derived from the gD gene of anHSV2 genome are included as experimental controls (the map from whichthe coordinates are based is found at GenBank Accession number K01408,HSVgD2 gene, and the HSVgD RNAs used in this study map to coordinates313-872).

[0158] Cells used in these studies are transfected with an expressionplasmid encoding a gene product known to interfere with the dsRNAinduced interferon response or with the PKR response, as describedabove. The cells are transfected with lipofectamine (Gibco-BRL) as atransfecting agent according to the manufacturer's instructions.

[0159] Two days after transfection, the cells are harvested and seededinto six-well plates and cultured to approximately 80 to 90% confluence.Cells are co-transfected with the T7 RNA polymerase expression plasmidand one of the RNA expression plasmids, such that one well of cellsreceives the T7 RNA polymerase expression plasmid and the gag sense RNAexpression plasmid; one well of cells receives the T7 RNA polymeraseexpression plasmid and the gag antisense RNA expression plasmid; onewell of cells receives the T7 RNA polymerase expression plasmid and thegag dsRNA expression plasmid; one well of cells receives the T7 RNApolymerase expression plasmid and the HSVgd sense RNA expressionplasmid; one well of cells receives the T7 RNA polymerase expressionplasmid and the HSVgd antisense RNA expression plasmid; and one well ofcells receives the T7 RNA polymerase expression plasmid and the HSVgDdsRNA expression plasmid. Transfection is again mediated throughlipofectamine (Gibco-BRL). There also is a control group of cellsreceiving no RNA. The cells are monitored for p24 synthesis over thecourse of several weeks. The cells are assayed both by measuring p24 inthe media of cells (using the p24 ELISA kit from Coulter, according tothe manufacturer's instructions) and by immunostaining fixed cells forp24 using a rabbit polyclonal anti-p24 sera and anti-rabbit IgG that isFITC conjugated (Sigma). None of the gD RNAs specifically shut down p24synthesis. The double stranded gag RNA significantly down regulates p24.The sense and antisense have only a modest effect on p24 synthesis andsome of the effect is predicted to be through the ability of the senseand antisense gag RNAs to generate low levels of dsRNA species.

EXAMPLE 10 Downregulation of PSA Expression in Human RhabdomyosarcomaCells Using Intracellular Expression of dsRNA

[0160] RD cells transiently expressing prostate specific antigen (PSA)were transfected with a T7 RNA polymerase expression vector and T7 RNAexpression vectors expressing PSA dsRNA, PSA sense RNA, PSA antisenseRNA, or control RNAs. The ability of the expressed RNAs to downregulatePSA expression was assessed, as described further below.

Creation of a Transient PSA Expression Line

[0161] The ability to downregulate expression of PSA following theexpression of a PSA specific double-stranded RNA (dsRNA) wasdemonstrated in a human rhabdomyosarcoma cell line. Since available PSAcell lines are difficult to work with (i.e., they are hard to transfect,and the cells tend to clump), a human cell line transiently expressingPSA was created. To create these cells, human rhabdomyosarcoma cellswere transiently transfected with a PSA plasmid-based expression vector,under conditions that result in transfection of greater than 95% of thecells. Transfection was mediated with lipofectamine transfecting reagent(Gibco-BRL) according to the manufacturer's instructions. Expression ofPSA was directed by the HCMV-IE promoter and the SV40 polyadenylationsignal (FIG. 2). PSA expression was measured in the supernatant oftransfected cells using a PSA ELISA kit (Oncogene Science Diagnostics,Cambridge, Mass.). No PSA was detected in the untransfected parentalcells while PSA was abundantly expressed in cells receiving the PSAexpression vector.

Downregulation of PSA Expression

[0162] The PSA expressing cell line was used as a model system withwhich to demonstrate the downregulation of PSA protein levels by PTGS.In these studies, plasmids encoding an approximately 600 nt sense RNA, a600 nt antisense RNA or a 600 nt dsRNA derived from a PSA cDNA were usedto transfect the PSA expressing cell line (FIG. 2). Expression of theRNAs was from a T7 RNA polymerase promoter(s) located at the 5′ end ofthe PSA sense strand, at the 5′ end of the PSA antisense strand, or atconverging positions at the 5′ ends of both the sense and antisensestrands respectively (FIG. 2). These encoded RNAs are not designed to beable to make protein (they do not have a cap, or a poly A tail).Transcription of the RNAs was catalyzed by T7 RNA polymerase, providedby a co-transfected T7 RNA expression plasmid. Control plasmidsexpressing similar sized sense RNA, antisense RNA, and dsRNA derivedfrom the glycoprotein D (gD) gene of an Herpes simplex 2 (HSV-2) genome,as described above were included as experimental controls.

[0163] Cells used in these studies can optionally be transfected with anexpression plasmid encoding a gene product known to interfere with thedsRNA induced interferon response or with the PKR response, as describedabove. The cells are transfected with lipofectamine (Gibco-BRL) as atransfecting agent according to the manufacturer's instructions.

[0164] Human rhabdomyosarcoma cells were seeded into six-well plates andcultured to approximately 80 to 90% confluence. The cells wereco-transfected with (A) the PSA expression plasmid; (B) one of the T7RNA expression plasmids; and (C) the T7 RNA polymerase expressionplasmid, such that all PSA expressing cells were transfected with the T7RNA polymerase expression plasmid and one of the following: the T7 sensePSA RNA expression construct, the T7 antisense PSA RNA expressionconstruct, the T7 dsRNA PSA expression construct, the sense HSVgD RNAexpression construct, the antisense HSVgD expression construct, or thedsRNA HSVgd expression construct. Cells received identical amounts ofthe PSA expression plasmid and the T7 RNA expression plasmid. Theamounts of the T7 RNA expression plasmids were also constant amongst thetransfected cells. Total DNA per transfection was held constant at 2.5μg DNA per one well of a six-well plate. In those transfections wherethere was no T7 RNA expression plasmid, an inert plasmid DNA was used asfiller DNA. Transfection was mediated by lipofectamine (Gibco-BRL)according to the manufacturer's instructions. There was also a controlgroup of untransfected cells, as well as an untreated PSA control groupof cells transfected with only the PSA expression plasmid in combinationwith the T7 RNA polymerase expression plasmid.

[0165] PSA-expressing cells that were not transfected with the T7 RNAexpression plasmid, as well as cells transfected with the T7 HSV2-gDRNAexpression plasmid all expressed PSA abundantly and at comparablelevels. Cells transfected with the sense, antisense, and ds PSA RNAexpression plasmids all exhibited varying degrees of inhibition of PSAexpression. A 5%-10% reduction in expression was seen in cellsexpressing the PSA sense RNA, a 50% reduction was seen in cellsexpressing the PSA antisense RNA and greater than 95% reduction was seenin the cells expressing the PSA dsRNA (FIG. 3). The inhibition was seenwithin two days after transfection and continued up until the last timepoint taken (one month later) at which point PSA levels were beginningto decline in the untreated cells and the experiment was terminated. Theuntreated PSA controls as well as cells transfected with the T7 HSV2-gDcontrol RNA expression plasmids all expressed PSA abundantly and atcomparable levels (FIG. 3), indicating the specificity of dsRNAeffectors to silence gene expression. During the one of month culture,cells were expanded into larger cultures at routine intervals.

[0166] Although the PSA specific dsRNA induced significant inhibition ofPSA expression, antisense and sense PSA RNAs also induced some level ofinhibition. Antisense PSA RNA has the potential to form dsRNA byannealing with PSA mRNA. Therefore the inhibition seen with antisenseRNA may be explained by both an antisense mechanism and a dsRNA inducedinhibition. A critical intracellular concentration of both antisense RNAand mRNA is required to generate dsRNA. Since much less dsRNA is made inthe antisense RNA expressing cells relative to those cells designed tomake dsRNA, a lesser inhibition of PSA in the antisense RNA expressingcells is expected if the threshold dsRNA levels required for efficientsilencing have not been reached in those cells. We have alsodemonstrated that a small amount of antisense RNA can be found in cellstransfected with our expression vectors (approximately 0.2% the amountof mRNA steady state levels). Antisense expression is presumably drivenby cryptic promoters on the non-coding plasmid DNA strand. The observedsense RNA inhibition could therefore also involve a dsRNA molecule. RNAfrom transfected but untreated cells could also be analyzed to determineif the low level expression of antisense RNAs in these cells results inthe production of detectable dsRNA species. Some low level expression ofPSA occurred in cells expressing PSA dsRNA. It is likely that somepercentage of cells did not take up the dsRNA expression cassette orthat the threshold levels of dsRNA were not reached in some cells. Nocellular toxicity was seen with any of the dsRNAs generated by the RNAexpression vectors suggesting that cytoplasmic expression of dsRNA doesnot induce the interferon response. In contrast, cell death is inducedwhen certain concentrations of in vitro produced dsRNA is delivered tocells via transfection with certain cationic lipids.

[0167] In summary, these results indicate that (i) PSA derived dsRNA ismuch more efficient than PSA antisense RNA in down-regulating PSAexpression, (ii) the down-regulation is sequence specific; only the PSAderived dsRNA and not the control HSV-2 derived dsRNA induceddown-regulation of PSA, and (iii) there is no toxicity associated withthe cytoplasmic expression of long (600 bp) dsRNA molecules.Additionally, these experiments are the first demonstration of dsRNAmediated down-regulation of gene expression in a human cell line.

EXAMPLE 11 Intracellular Expression of dsRNA does not Induce the Type 1Interferon Response (RNA stress response).

[0168] Human rhabdomyosarcoma (RD) cells were transfected with variousdsRNA expression vectors such that dsRNA was transcribed in thetransfected cells as described in Example 10. Transcription of dsRNAoccurred in the cells within 24 hours after transfection and continuedfor the duration of the thirty day experiment. Cells and thesupernatants from transfected cells were analyzed during the course ofthe experiment for any evidence of RNA stress response induction. Noevidence of RNA stress response induction by intracellular expresseddsRNA was observed. RD cells have been shown by us to be responsive totype 1 interferon, both alpha and beta, and thus RD cells are capable ofmounting an RNA stress response. In addition, positive controls wereincluded in these experiments. A positive control for these experimentsis a method of delivering dsRNA which induces the RNA stress response.All positive controls induced the RNA stress response. These experimentsare described further below.

Assays Performed to Identify RNA Stress Response Induction

[0169] The following assays were performed to measure the induction ofan RNA stress response: TUNEL assay to detect apoptotic cells, ELISAassays to detect the induction of alpha, beta and gamma interferon,ribosomal RNA fragmentation analysis to detect activation of 2′5′OAS,measurement of phosphorylated eIF2a as an indicator of PKR (proteinkinase RNA inducible) activation, proliferation assays to detect changesin cellular proliferation, and microscopic analysis of cells to identifycellular cytopathic effects. Apoptosis, interferon induction, 2′5′ OASactivation, PKR activation, anti-proliferative responses, and cytopathiceffects are all indicators for the RNA stress response pathway.

Transfection of Cells

[0170] Approximately 7×10⁵ RD cells were seeded into individual wells ofsix-well plates. Cells were transfected when they reached about 90%confluency. Cells were transfected with a T7 RNA polymerase expressionconstruct and a T7 dsRNA expression construct. The T7 dsRNA expressionconstructs encode converging T7 promoters located on either side of a600 bp sequence (FIG. 2). Controls included cells transfected with theT7 RNA expression construct alone so that no dsRNA is made in thesecells. Total DNA per transfection was held constant at 2.5 μg DNA perone well of a six-well plate. When the T7 RNA polymerase and T7 dsRNAexpression vectors were both used, 1.25 μg of each DNA was used pertransfection. In those transfections where there was no T7 dsRNAexpression construct, inert filler DNA was used to bring the total DNAto 2.5 μg per transfection. Transfection was mediated usingLipofectamine (InVitrogen) according to the manufacturer's directions.The positive control transfections included poly(I)(C) RNA and in vitrotranscribed dsRNA of 600 bp that were both complexed with Lipofectamineand transfected into cells. The cells were transfected with in vitrotranscribed ssRNA complexed with Lipofectamine. 2.5 μg of each RNA wasused per transfection. Other controls included untreated cells. Cellswere kept in culture for one month by expanding into larger flasks asthe cell numbers increased.

ELISA Assays

[0171] Supernatants were removed from the transfected and untreatedcells at time points of 1, 2, 7, 17, and 48 hours and every several daysfor up to one month after the 48 hour time point. Collected supernatantswere stored at −80° C. until they were analyzed for the presence ofalpha, beta, and gamma interferon using commercially available ELISAkits. The Interferon-alpha ELISA kit was obtained from ENDOGEN(Rockford, Ill.), the Interferon-Beta ELISA kit was obtained from RD 1(Flanders, N.J.), and the Interferon-gamma ELISA kit was obtained fromR&D Systems (Minneapolois, Minn.). ELISAs were all performed accordingto the manufacturer's directions. Alpha, beta, and gamma interferon werenot detected at increased levels in cells expressing intracellular dsRNAcompared to the corresponding levels in untreated cells. However,considerable levels of beta interferon were found in cells transfectedwith poly (I)(C) or with in vitro transcribed dsRNA and ssRNA. Alpha andbeta interferon induction are associated with induction of the RNAstress response.

TUNEL Assay

[0172] Cells were stained for the presence of apoptotic nuclei using acommercially available kit, TdT FragEL, DNA Fragmentation Detection Kit,In Situ Apoptosis Assay from Oncogene (Boston, Mass.). Cells werestained according the manufacturer's directions. Cells were stained at 2hours, 7 hours, 17 hours, 2 days, 3 days, 4 days, and 5 days aftertransfection. There was no evidence of apoptosis induced byintracellular expressed dsRNA at any of the time points analyzed.However, the majority of cells transfected with poly (I)(C) or with thein vitro transcribed dsRNA were apoptotic by 17 hours aftertransfection. No evidence of apoptosis was observed in the untreatedcells or in cells transfected with ssRNA. Apoptosis is an end result ofthe induction of the RNA stress response pathway.

2′5′OAS Activation

[0173] The activation of 2′5′OAS was determined by performing ribosomalRNA fragmentation analysis. Briefly, following transfection, total RNAwas extracted from cells using standard procedures. RNA was extracted atthe following time points: 2 hours, 7 hours, 17 hours, 48 hours, 3 days,4 days, and 5 days after transfection. 5-10 μg RNA was analyzed for eachsample. RNA samples were first denatured in formaldehyde/formamide RNAsample buffer at 65° C. for 10 minutes prior to being electrophoresedthrough 0.5×TBE agarose gels. Ribosomal RNA was visualized by stainingwith ethidium bromide followed by ultraviolet transillumination.Ribosomal RNA fragmentation was observed in cells transfected with poly(I)(C) and with the in vitro transcribed dsRNA. No fragmentation wasobserved in the untreated control cells, cells transfected with ssRNA,or in cells expressing intracellular dsRNA. These results indicate that2′5′OAS was not activated by dsRNA when it was made intracellularly.2′5′OAS activation is associated with induction /activation of the RNAstress response pathway.

PKR Activation

[0174] The activation of PKR was determined by measuring the levels ofeIF2alpha phosphorylation. Briefly, cells were lysed at various timesafter transfection (2 hours, 7 hours, 19 hours, 48 hours, 3 days, 4days, and 5 days after transfection) and analyzed for levels ofphosphorylated and non-phosphorylated eIF2 alpha. The protocol forlysing cells can be found in the following reference: Zhang F. et al.,J. Biol. Chem. 276(27):24946-58, 2001. This analysis was performed asdescribed for detecting PKR phosphorylation except that antibodiesspecific for phosphorylated and non-phosphorylated eIF2alpha were used.These antibodies are available from Cell Signaling Technology (Beverly,Mass.).

Cytopathic Effect and Antiproliferative Responses

[0175] Cytopathic effect was assayed by analyzing cells microscopicallyusing a light microscope. Cells were analyzed at daily intervalsthroughout the course of the experiment. Cytopathic effect is defined asany or all of the following: cells detaching from surface of well/flask,cells rounding up, an increased number of vacuoles in transfected cellswith respect to the control untreated cells, or differences inmorphology of cells with respect to the untreated control cells. Nocytopathic effect was seen in those cells expressing dsRNAintracellularly. Severe cytopathic effects were seen in cellstransfected with Poly (I)(C) or with dsRNA made in vitro. Cytopathiceffect is associated with the RNA stress response.

[0176] Antiproliferative responses were assayed by measuring thedivision rate of cells. The division rate is determined by counting cellnumbers using standard procedures. Cells were counted every few days forthe duration of the experiment. No antiproliferative responses were seenin cells expressing dsRNA intracellulary. Antiproliferative responsesare associated with the RNA stress response.

Summary of Results

[0177] The results of the above assays indicate that intracellularexpression of dsRNA does not induce the RNA stress response. The cellsthat were used for these experiments were competent for RNA stressresponse induction as was demonstrated by the ability of cationic lipidcomplexed poly(I)(C) and in vitro transcribed RNA to induce/activate alltested components of this response. In addition, the cells were found tobe responsive to exogenously added interferon. These results imply thatthe cells used for these experiments are not defective in their abilityto mount an RNA stress response and therefore can be used as predictorsfor other cells, both in cell culture and in vivo in animal models. Thismethod described here, which does not induce the interferon stressresponse, has been found to induce PTGS. This method therefore providesa method to induce PTGS without inducing an undesired RNA stressresponse.

[0178] Although these results were generated using a vector thatutilizes a T7 transcription system and therefore expresses dsRNA in thecytoplasm, the vector system can be changed to other systems thatexpress dsRNA intracellularly. Similar results are expected with theseexpression systems. These systems include, but are not limited to,systems that express dsRNA or hairpin RNAs in the nucleus, in thenucleus followed by transport of the RNAs to the cytoplasm, or in thecytoplasm using non-T7 RNA polymerase based expression systems.

EXAMPLE 12 Optimization of the Concentrations and Relative Ratios of invitro or in vivo Produced dsRNA and Delivery Agent

[0179] The optimal concentrations and ratios of dsRNA to a deliveryagent such as a cationic lipid, cationic surfactant, or local anestheticcan be readily determined to achieve low toxicity and to efficientlyinduce gene silencing using in vitro or in vivo produced dsRNA.

Summary of Factors Effecting Nucleic Acid/cationic Lipid Interactions

[0180] Cationic lipid DNA interactions are electrostatic. Electrostaticinteractions are highly influenced by the ionic components of themedium. The ability to form stable complexes is also dependent upon theintermolecular interactions between the lipid molecules. At lowconcentrations, certain inter-lipid interactions are preferred; athigher lipid concentrations, rapid condensates are formed due to higherorder interactions. Although local interactions are similar in both ofthese instances (e.g., phosphoryl groups in the DNA and the chargedcationic head group), the long range and inter-lipid interactions aresubstantially different. Similarly, structurally diverse variants can beobtained simply by changing the charge ratio of the complex by mixingvarying amounts of cationic lipid with fixed concentrations of thenucleic acid or vice versa. This variation in the structure of thecomplexes is evidenced by altered physical properties of the complexes(e.g., differences in octanol partitioning, mobility on densitygradients, charge density of the particle, particle size, andtransfectability of cells in culture and in vivo) (Pachuk et al. DNAVaccines—Challenges in Delivery, Current Opinion in MolecularTherapeutics, 2(2) 188-198, 2000 and Pachuk et al, BBA, 1468, 20-30,(2000)). Furthermore, different lipids, local anesthetics, andsurfactants differ in their interactions between themselves, andtherefore novel complexes can be formed with differing biophysicalproperties by using different lipids singularly or in combination. Foreach cell type, the following titration can be carried out to determinethe optimal ratio and concentrations that result in complexes that donot induce the stress response or interferon response. At several ofthese concentrations PTGS is predicted to be induced; however, PTGS ismost readily observed under conditions that result in highly diminishedcytotoxicity.

Complex Formation

[0181] dsRNA is either produced by in vitro transcription using the T7promoter and polymerase or another RNA polymerase, such as an E. coliRNA polymerase. dsRNA can also be produced in an organism or cell usingendogenous polymerases.

[0182] Concentrations of dsRNA, such as PSA-specific dsRNA, are variedfrom 1 pg to 10 μg. In some instances, 150 ng of a plasmid that encodesa reporter of interest (PSA) to be silenced may be comixed at aconcentration between 10 ng and 10 μg. The concentration of cationiclipid, cationic surfactant, local anesthetic, or any other transfectionfacilitating agent that interacts with the nucleic acidelectrostatically are varied at each of the dsRNA concentrations toyield charge ratios of 0.1 to 1000 (positive/negative) (i.e., the ratioof positive charge from lipids or other delivery agents to negativecharge from DNA or RNA). The complexes are prepared in water or inbuffer (e.g., phosphate, HEPES, citrate, Tris-HCl, Tris-glycine, malate,etc. at pH values that range from 4.0 to 8.5), may contain salt (e.g.,1-250 mM), and may contain glycerol, sucrose, trehalose, xylose, orother sugars (e.g., mono-, di-, or polysaccharide). The mixture isallowed to sit at room temperature, desirably for 30 minutes, and may bestored indefinitely. The complexes are premixed in serum free media. Thenucleic acid and the transfecting reagent may be mixed either throughdirect addition or through a slow mixing process, such as across adialyzing membrane or through the use of a microporous particle or adevice that brings the two solutions together at a slow rate and at lowconcentrations. In some instances, the two interacting components aremixed at low concentrations, and the final complex is concentrated usinga diafilteration or any other concentrating device. Alternatively, ifthe complexes are formed at high concentrations of either or both of theinteracting components, the complexes may be diluted to form an idealtransfection mixture.

Transfection Protocol and Analysis of dsRNA Stress Response

[0183] Complexes are added to cells that are ˜60-80% confluent in serumfree media. The complexes are incubated for various times (e.g., 10minutes to 24 hours) with the cells at 37° C. and diluted with serumcontaining media or washed and replated in serum free media. The cellsare monitored for toxicity and analyzed at various times for signs ofdsRNA response (e.g., TUNNEL assay to detect nicked DNA, phosphorylationof EIF2alpah, induction and activation of 2′5′ OAS, or interferon-alphaand -beta). Transfection conditions that result in less than 50%, 25%,10%, or 1% cytotoxicity or that result in a less than 20, 10, 5, 2, or1.5-fold induction of a stress response are analyzed to determine ifPTGS was efficiently induced.

Determining Induction of PTGS

[0184] PSA protein levels are determined in cell culture media usingstandard methods. The data is normalized to the number of live cells inculture to determine the concentrations required to induce PTGS.

Results

[0185] Using the above method, cationic lipid complexes of dsRNA inducedtoxicity at certain ranges. With lipofectamine as the cationic lipid,positive to negative charge ratios greater than 10 did not produce anydetectable toxicity at any of the concentrations of dsRNA tested andinduced a high level of PTGS, resulting in highly decreased levels ofPSA in the culture medium. The RNA concentration ranges tested were 1 pgto 100 ng with a constant amount of lipofectamine (10 uL of a 2 mg/mLsolution from GIBCO-BRL Life Technologies, Bethesda, Md.).

EXAMPLE 13 Method to Avoid dsRNA Mediated Activation of the RNA StressResponse Pathway

[0186] One or more components of the RNA stress response pathway can bemutated or inactivated to avoid induction/activation of the component(s)by dsRNA that is delivered to the cell or animal for the purpose ofinducing PTGS. These components, such as those illustrated in FIG. 4,can be knocked out singularly or in combination.

[0187] Various standard methods can be used to knockout components ofthe RNA stress response pathway, such as PKR, human beta interferonAccession No. M25460), and/or 2′5′OAS (Accession No. NM_(—)003733).Alternatively or additionally, one or more interferon response element(IRE) sequences can be mutated or deleted using a knockout constructdesigned based on the IRE consensus sequence (Ghislain, et al., JInterferon Cytokine Res. 2001 June 21(6):379-88.), and/or one or moretranscription factors that bind IRE sequences, such as STAT1 (Accessionnumber XM_(—)010893), can be mutated or deleted. These methods includethe use of antisense DNA/RNA, ribozymes, or targeted gene knockouttechnology mediated by homologous recombination. One skilled in the artis able to design the appropriate antisense sequences, ribozymes, andvectors for targeted knockouts. For example, targeted knockouts may beprepared by any of the following standard methods: Shibata et al., ProcNatl Acad Sci U S A. 2001 July 17;98(15):8425-32. Review., Muyrers etal., Trends Biochem Sci. 2001 May;26(5):325-31., Paul et al, Mutat Res.2001 June 5;486(1):11-9., Shcherbakova et al., Mutat Res. 2000 February16;459(1):65-71., Lantsov. Ideal gene therapy: approaches and prospectsMol Biol (Mosk). 1994 May-June;28(3):485-95., in Gene Transfer andExpression—A Laboratory Manual editor: Michael Kriegler, Publisher—WHFreeman & Co, New York, N.Y., pages 56-60, 1990).

[0188] Knockout cells can be readily identified either through the useof an antibiotic resistance marker which when transferred to thechromosome confers resistance to the cell or through the use of dsRNAitself. In particular, dsRNA (e.g., a high concentration of dsRNA)induces apoptosis in wild-type cells while mutant cells survive dsRNAtreatment because they cannot mount a stress response. Yet anotherapproach involves performing the dsRNA-induced PTGS experiment in thepresence of large concentrations of IRE (dsDNA) oligo, which is expectedto titrate activated STAT proteins. These oligos can be deliveredintracerllularly using transfecting agents or electroporation.

[0189] In another method of preventing the interferon response, cells(e.g., RD cells) are transfected with a T7 RNA polymerase expressionvector and a T7 dsRNA expression vector encoding dsRNA homologous to thehuman protein kinase PKR cDNA (accession number M35663) or homologous tothe coding sequence of any other component in the RNA stress responsepathway. In one particular example, dsRNA corresponding to nucleotides190-2000 is encoded by the T7dsRNA expression vector. The expressionvectors are similar to those described in Example 10 and shown in FIG.2, except that the dsRNA encoding sequence is derived from the humanprotein kinase PKR cDNA. Transfection in RD cells is performed asdescribed in Example 10. Within 2-5 days posttransfection, the cells arefunctionally PKR negative.

Other Embodiments

[0190] From the foregoing description, it will be apparent thatvariations and modifications may be made to the invention describedherein to adopt it to various usages and conditions. Such embodimentsare also within the scope of the following claims.

[0191] All publication, patents, and patent applications mentioned inthis specification are herein incorporated by reference to the sameextent as if each individual publication, patent, or patent applicationwas specifically and individually indicated to be incorporated byreference.

What is claimed is:
 1. A method for identifying a nucleic acid thatmodulates the function of a vertebrate cell, said method comprising thesteps of: (a) transforming a population of vertebrate cells with adouble stranded RNA expression library, wherein at least two cells ofsaid population of cells are each transformed with a different nucleicacid from said double stranded RNA expression library, wherein saidtransformed nucleic acid is capable of forming double stranded RNA, andwherein said transformation and said formation of double stranded RNAare carried out under conditions that inhibit or prevent an interferonresponse or a double stranded RNA stress response; (b) selecting avertebrate cell in which said nucleic acid is expressed in said cell;and (c) assaying for a modulation in the function of said cell, whereinsaid modulation identifies a nucleic acid that modulates the function ofsaid vertebrate cell.
 2. A method for identifying a nucleic acid thatmodulates expression of a target nucleic acid in a vertebrate cell, saidmethod comprising the steps of: (a) transforming a population ofvertebrate cells with a double stranded RNA expression library, whereinat least two cells of said population of cells are each transformed witha different nucleic acid from said double stranded RNA expressionlibrary, wherein said transformed nucleic acid is capable of formingdouble stranded RNA, and wherein said transformation and said formationof double stranded RNA are carried out under conditions that inhibit orprevent an interferon response or a double stranded RNA stress response;(b) selecting a vertebrate cell in which said nucleic acid is expressedin said cell; and (c) assaying for a modulation in the expression of atarget nucleic acid in said cell, wherein said modulation identifies anucleic acid that modulates expression of said target nucleic acid.
 3. Amethod for identifying a nucleic acid that modulates the biologicalactivity of a target polypeptide in a vertebrate cell, said methodcomprising the steps of: (a) transforming a population of vertebratecells with a double stranded RNA expression library, wherein at leasttwo cells of said population of cells are each transformed with adifferent nucleic acid from said double stranded RNA expression library,wherein said transformed nucleic acid is capable of forming doublestranded RNA, and wherein said transformation and said formation ofdouble stranded RNA are carried out under conditions that inhibit orprevent an interferon response or a double stranded RNA stress response;(b) selecting for a vertebrate cell in which said nucleic acid isexpressed in said cell; and (c) assaying for a modulation in thebiological activity of a target polypeptide in said cell, wherein saidmodulation identifies a nucleic acid that modulates the biologicalactivity of said target polypeptide.
 4. The method of claim 1, whereinat most one nucleic acid is stably integrated into a chromosome of eachcell.
 5. The method of claim 1, said method further comprising: (d)identifying said nucleic acid by amplifying said nucleic acid andsequencing said amplified nucleic acid.
 6. The method of claim 1,wherein said double stranded RNA expression library comprises cDNAsderived from said cells.
 7. The method of claim 1, wherein said doublestranded RNA expression library comprises randomized nucleic acids. 8.The method of claim 1, wherein said double stranded RNA expressionlibrary is a nuclear double stranded RNA expression library.
 9. Themethod of claim 1, wherein said double stranded RNA expression libraryis a cytoplasmic double stranded RNA expression library.
 10. The methodof claim 1, wherein said cell is a mammalian cell.
 11. The method ofclaim 10, wherein said cell is a human cell.
 12. The method of claim 10,wherein said cell is selected from the group consisting of a cancercell, a cell of the immune system, a stem cell, a neuronal cell, amuscle cell, and an adipocyte.
 13. The method of claim 1, wherein saidnucleic acid is contained in a vector.
 14. The method of claim 13,wherein said vector comprises an RNA polymerase II promoter, an RNApolymerase I promoter, an RNA polymerase III promoter, or amitochondrial promoter.
 15. The method of claim 13, wherein the sensestrand and the anti-sense strand of said double stranded RNA aretranscribed from the same nucleic acid using two convergent promoters.16. The method of claim 13, wherein said nucleic acid comprises aninverted repeat such that upon transcription said nucleic acid forms adouble stranded RNA.
 17. The method of claim 1, wherein said assayingcomprises measuring an event selected from the group consisting of cellmotility, apoptosis, cell growth, cell invasion, vascularization, cellcycle events, cell differentiation, cell dedifferentiation, neuronalcell regeneration, and the ability of a cell to support viralreplication.
 18. The method of claim 1, wherein said double stranded RNAis between 5 and 100 nucleotides in length, inclusive.
 19. The method ofclaim 1, wherein said double stranded RNA is at least 100 nucleotides inlength.
 20. The method of claim 19, wherein said double stranded RNA isat least 250 nucleotides in length.
 21. The method of claim 20, whereinsaid double stranded RNA is at least 500 nucleotides in length.
 22. Themethod of claim 21, wherein said double stranded RNA is at least 1000nucleotides in length.
 23. A method for identifying a nucleic acid thatmodulates the function of a vertebrate cell, said method comprising thesteps of: (a) transforming a population of vertebrate cells with adouble stranded RNA under conditions that inhibit or prevent aninterferon response or a double stranded RNA stress response; (b)selecting a vertebrate cell in which said double stranded RNA isexpressed; and (c) assaying for a modulation in the function of saidcell, wherein said modulation identifies a nucleic acid that modulatesthe function of said cell.
 24. A method for identifying a nucleic acidthat modulates expression of a target nucleic acid in a vertebrate cell,said method comprising the steps of: (a) transforming a population ofvertebrate cells with a double stranded RNA under conditions thatinhibit or prevent an interferon response or a double stranded RNAstress response; (b) selecting a vertebrate cell in which said doublestranded RNA is expressed; and (c) assaying for a modulation in theexpression of a target nucleic acid in said cell, wherein saidmodulation identifies a nucleic acid that modulates expression of saidtarget nucleic acid.
 25. A method for identifying a nucleic acid thatmodulates the biological activity of a target polypeptide in avertebrate cell, said method comprising the steps of: (a) transforming apopulation of vertebrate cells with a double stranded RNA underconditions that inhibit or prevent an interferon response or a doublestranded RNA stress response; (b) selecting a vertebrate cell in whichsaid double stranded RNA is expressed; and (c) assaying for a modulationin the biological activity of a target polypeptide in said cell, whereinsaid modulation identifies a nucleic acid that modulates the biologicalactivity of said target polypeptide.
 26. The method of claim 23, saidmethod further comprising: (d) identifying said nucleic acid byamplifying said nucleic acid and sequencing said amplified nucleic acid.27. The method of claim 23, wherein said double stranded RNA is derivedfrom said cells.
 28. The method of claim 23, wherein said doublestranded RNA is between 5 and 100 nucleotides in length, inclusive. 29.The method of claim 23, wherein said double stranded RNA is at least 100nucleotides in length.
 30. The method of claim 29, wherein said doublestranded RNA is at least 250 nucleotides in length.
 31. The method ofclaim 30, wherein said double stranded RNA is at least 500 nucleotidesin length.
 32. The method of claim 31, wherein said double stranded RNAis at least 1000 nucleotides in length.
 33. The method of claim 23,wherein each cell is transformed with at most one double stranded RNA.34. The method of claim 23, wherein said cell is a mammalian cell. 35.The method of claim 34, wherein said cell is a human cell.
 36. Themethod of claim 23, wherein said population is transformed with at least100 different double stranded RNAs.